Tests for the rapid evaluation of ischemic states and kits

ABSTRACT

The present invention relates to rapid methods for the detection of ischemic states and to kits for use in such methods. Provided for is a rapid method of testing for and quantifying ischemia based upon methods of detecting and quantifying the existence of an alteration of the serum protein albumin which occurs following an ischemic event; methods for detecting and quantifying this alteration include evaluating and quantifying the cobalt binding capacity of circulating albumin, analysis and measurement of the ability of serum albumin to bind exogenous cobalt, detection and measurement of the presence of endogenous copper in a purified albumin sample and use of an immunological assay specific to the altered form of serum albumin which occurs following an ischemic event. Also taught by the present invention is the detection and measurement of an ischemic event by measuring albumin N-terminal derivatives that arise following an ischemic event, including truncated albumin species lacking one to four N-terminal amino acids or albumin with an acetylated N-terminal Asp residue.

The subject application is a divisional of U.S. Ser. No. 09/806,247,filed Mar. 27, 2001 now abandoned; which is a 35 USC 371 ofPCT/US99/22905, filed Oct. 1, 1999, which is a continuation-in-part ofU.S. Ser. No. 09/165,581, filed Oct. 2, 1998, now U.S. Pat. No.6,492,179; and a continuation-in-part of U.S. Ser. No. 09/165,926, filedOct. 2, 1998, now U.S. Pat. No. 6,461,875. The foregoing cases are allincorporated by reference in their entirety. PCT/US99/22905 also claimedthe benefit of priority from U.S. Ser. No. 60/115,392, filed Jan. 11,1999, now expired, and U.S. Ser. No. 60/102,738, filed Oct. 2, 1998, nowexpired.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to rapid methods for the detection ofischemic states and to kits for use in such methods. More particularly,the invention relates to the measurement of a bound specific transitionelement to human serum albumin or the measurement of albumin N-terminalderivatives to determine the presence or absence of ischemia.

2. Discussion of the Background

Ischemia is the leading cause of illness and disability in the world.Ischemia is a deficiency of oxygen in a part of the body causingmetabolic changes, usually temporary, which can be due to a constrictionor an obstruction in the blood vessel supplying that part. The two mostcommon forms of ischemia are cardiovascular and cerebrovascular.Cardiovascular ischemia, in which the body's capacity to provide oxygento the heart is diminished, is the leading cause of illness and death inthe United States. Cerebral ischemia is a precursor to cerebrovascularaccident (stroke) which is the third leading cause of death in theUnited States.

The continuum of ischemic disease includes five conditions: (1) elevatedblood levels of cholesterol and other blood lipids; (2) subsequentnarrowing of the arteries; (3) reduced blood flow to a body organ (as aresult of arterial narrowing); (4) cellular damage to an organ caused bya lack of oxygen; (5) death of organ tissue caused by sustained oxygendeprivation. Stages three through five are collectively referred to as“ischemic disease,” while stages one and two are considered itsprecursors.

Together, cardiovascular and cerebrovascular disease accounted for954,720 deaths in the U.S. in 1994. Furthermore, more than 20% of thepopulation has some form of cardiovascular disease. In 1998, as many as1.5 million Americans will have a new or recurrent heart attack, andabout 33% of them will die. Additionally, as many as 3 to 4 millionAmericans suffer from what is referred to as “silent ischemia.” This isa condition where no clinical symptoms of ischemic heart disease arepresent. There is currently a pressing need for the development andutilization of blood tests able to detect injury to the heart muscle andcoronary arteries. Successful treatment of cardiac events dependslargely on detecting and reacting to the presence of cardiac ischemia intime to minimize damage. Cardiac enzymes, specifically the creatinekinase isoenzyme (CK-MB), and cardiac markers, specifically the TroponinI and T biochemical markers, are utilized for diagnosing heart muscleinjury. However, these enzymes and markers are incapable of detectingthe existence of an ischemic state in a patient prior to myocardialinfarction and resulting cell necrosis (death of cell). Additionally,these enzymes and markers do not show a measurable increase untilseveral hours after an ischemic event. For instance, CK-MB, the earlierevident of the two, does not shows a measurable increase above normal ina person's blood test until about four to six hours after the beginningof a heart attack and does not reach peak blood level until about 18hours after such an event. Thus, the primary shortcoming of usingcardiac markers for diagnosis of ischemic states is that these markersare only detectable after heart tissue has been irreversibly damaged.

There currently are no tests available which allow diagnosis of theexistence of ischemia in patients prior to tissue necrosis. A pressingrequirement for emergency medicine physicians who treat chest pain andstroke symptoms is for a diagnostic test that would enable them todefinitively “rule out” myocardial infarction, stroke, and otheremergent forms of ischemia. A need exists for a method for immediate andrapid distinction between ischemic and non-ischemic events, particularlyin patients undergoing acute cardiac-type symptoms. The presentinvention provides such a means.

A broader array of diagnostic tests are available for diagnosis ofischemia in patients with non-acute symptoms. The EKG exercise stresstest is commonly used as an initial screen for cardiac ischemia, but islimited by its accuracy rates of only 25-50%. Coronary angiography, aninvasive procedure that detects narrowing in the arteries with 90-95%accuracy, is also utilized. Another commonly used diagnostic test is thethallium exercise stress test, which requires injection of radioactivedye and serial tests conducted four hours apart. The present invention,however, has the advantage over the known methods of diagnosis in thatit provides equivalent or better accuracy at far lower costs anddecreased risk and inconvenience to the patient. The present inventionprovides specificity and sensitivity levels of 75-95%, which are farmore accurate than the EKG exercise stress test and comparable inaccuracy to current diagnostic standards. Furthermore, the presentinvention presents a significant time advantage and is cheaper thancompeting methods of diagnosis by a factor of at least 15 to 1.

It is known that immediately following an ischemic event, proteins(enzymes) are released into the blood. Well known proteins releasedafter an ischemic heart event include creatine kinase (CK), serumglutamic oxalacetic transaminase (SGOT) and lactic dehydrogenase (LDH).One well known method of evaluating the occurrence of past ischemicheart events is the detection of these proteins in a patient's blood.U.S. Pat. No. 4,492,753 relates to a similar method of assessing therisk of future ischemic heart events. However, injured heart tissuereleases proteins to the bloodstream after both ischemic andnon-ischemic events. For instance, patients undergoing non-cardiacsurgery may experience perioperative ischemia. Electro-cardiograms ofthese patients show ST-segment shifts with an ischemic cause which arehighly correlated with the incidence of postoperative adverse cardiacevents. However, ST-segment shifts also occur in the absence ofischemia; therefore, electrocardiogram testing does not distinguishischemic from non-ischemic events. The present invention provides ameans for distinguishing perioperative ischemia from ischemia caused by,among other things, myocardial infarctions and progressive coronaryartery disease.

SUMMARY OF THE INVENTION

The present need for rapid, immediate and continuous detection ofischemic states is met by the present invention. Specifically, thepresent invention provides for rapid methods of testing for theexistence of and quantifying ischemia based upon methods of detectingand quantifying the existence of an alteration of the serum proteinalbumin which occurs following an ischemic event. Preferred methods ofthe present invention for detecting and quantifying this alterationinclude evaluating and quantifying the metal binding capacity ofalbumin, analysis and measurement of the ability of serum albumin tobind exogenous metal, detection and measurement of the presence ofendogenous copper in a purified albumin sample, use of an immunologicalassay specific to albumin-metal complexes, and detection and measurementof albumin N-terminal derivatives that arise following an ischemicevent. Also taught by the present invention is the use of the compoundAsp-Ala-His-Lys-R, wherein R is any chemical group capable of beingdetected when bound to a metal ion that binds to the N-terminus ofnaturally occurring human albumin, for detection and quantitation of anischemic event.

Advantages and embodiments of the invention include a method forruling-out the existence of an ischemic state or event in a patient; amethod for detecting the existence of asymptomatic ischemia; a methodfor evaluating patients with angina to rule-out the recent occurrence ofan ischemic event; an immediate method for evaluation of patientssuffering from chest pain to detect the recent occurrence of amyocardial infarction; a method for evaluation of patients sufferingfrom stroke-like signs and symptoms to detect the occurrence of a strokeand to distinguish between the occurrence of an ischemic stroke and ahemorrhagic stroke; a rapid method for supplementingelectrocardiographic results in determining the occurrence of trueischemic events; a method for detecting the occurrence of a trueischemic event in a patient undergoing surgery; a method for evaluatingthe progression of patients with known ischemic conditions; a method forcomparing levels of ischemia in patients at rest and during exercise; amethod for assessing the efficacy of an angioplasty procedure; a methodfor assessing the efficacy of thrombolytic drug therapy; a method forassessing the patency of an in-situ coronary stent; and, a method fordetecting in a pregnant woman the occurrence of placental insufficiency.

Additional advantages, applications, embodiments and variants of theinvention are included in the Detailed Description of the Invention andExamples sections.

As used herein, the term “ischemic event,” and “ischemic state” meanthat the patient has experienced a local and/or temporary ischemia dueto partial or total obstruction of the blood circulation to an organ.Additionally, the following abbreviations are utilized herein to referto the following amino acids:

Amino acid Three-letter abbreviation Single-letter notation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine or AsxB aspartic acid Cysteine Cys C Glutamine Gln Q Glutamic acid Glu EGlutamine or Glx Z glutamic acid Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

A separate test method for ischemia was described by a common inventorin U.S. Pat. Nos. 5,227,307 and 5,290,519 to Bar-Or et al., hereinincorporated by reference in their entirety. Also incorporated herein intheir entireties by reference are the following commonly assignedapplications: U.S. Ser. No. 09/165,926, filed Oct. 2, 1998, now U.S.Pat. No. 6,4461,875; U.S. Ser. No. 09/165,581, filed Oct. 2, 1998, nowU.S. Pat. No. 6,492,179; and U.S. Ser. No. 60/115,392, filed Jan. 11,1999, now expired.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-3 illustrate kits useful in carrying out the derivativeembodiment of the subject invention.

FIG. 4 shows selected regions of the ¹H-NMR spectra (500 MHz, 10% D₂O inH₂O, 300K) showing the Ala resonances (Ala-2 and Ala-8) of theoctapeptide (Asp-Ala-His-Lys-Ser-Glu-Val-Ala, residues 1-8 of SEQ. ID.NO. 1) (a) free of any metal, with a Lys-4 methylene resonance appearingbetween the doublets, (b) with 0.5 equiv. of NiCl₂ added, (c) with 1.0equiv. of NiCl₂ added, (d) with 0.5 equiv. of CoCl₂ added, and (e) with1.0 equiv. of CoCl₂ added.

FIGS. 5A and 5B are ultraviolet spectra for non-acetylated Pep-12(Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys, residues 1-12 of SEQ.ID. NO. 1) and acetylated Pep-12, respectively.

FIGS. 6A and 6B are ultraviolet spectra for non-acetylated Pep-12 andacetylated Pep-12 each with CoCl₂, respectively.

FIG. 7 provides spectral analysis of five solutions of increasingproportions of acetylated Pep-12 to non-acetylated Pep-12 with effect oncobalt binding as reflected by a shift in absorbance from 220 to 230.

FIGS. 8A and 8B are U.V. spectra of Pep-12 and acetylated Pep-12,respectively, mixed first with CuCl₂ and then with CoCl₂.

FIG. 9 is the U.V. spectra of acetylated Pep-8(Asp-Ala-His-Lys-Ser-Glu-Val-Ala, residues 1-8 of SEQ. ID. NO. 1) whichdid not shift upon addition of cobalt.

FIGS. 10A-D are the ¹H-NMR spectra of Peptide 1(Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys, residues 1-12 of SEQ.ID. NO. 1) which shows the methyl signals of the two Ala residues atpositions 2 and 8 as titrated by NiCl₂. FIG. 10A is Peptide 1 at pH2.55, while 10B is at pH 7.33. FIG. 10C is the spectra at pH 7.30 with0.3 equiv. NiCl₂, and FIG. 10D is pH 7.33 at about ˜1 equiv. NiCl₂.

FIGS. 11A-D are the ¹H-NMR spectra of Peptide 1(Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys, residues 1-12 of SEQ.ID. NO. 1) which shows the methyl signals of the two Ala residues atpositions 2 and 8 as titrated by CoCl₂. FIG. 11A is Peptide 1 at pH2.56, while 11B is at pH 7.45. FIG. 11C is the spectra at pH 7.11 withabout ˜0.5 equiv. CoCl₂, and FIG. 11D is pH 7.68 at about ˜1 equiv.CoCl₂.

FIGS. 12A-D are the ¹H-NMR spectra of Peptide 1(Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys, residues 1-12 of SEQ.ID. NO. 1) which shows the methyl signals of the two Ala residues atpositions 2 and 8 as titrated by CuSO₄. FIG. 12A is Peptide 1 at pH2.56, while 12B is at pH 7.54. FIG. 12C is the spectra at pH 7.24 with˜0.5 equiv. CuSO₄, and FIG. 12D is pH 7.27 at about ˜1 equiv. CuSO₄.

FIGS. 13A-D are the ¹H-NMR spectra of Peptide 2, which is theacetylated-Asp version of Peptide 1. FIG. 13A is Peptide 2 at pH 2.63.FIG. 13B is Peptide 2 at pH 7.36. FIG. 13C is Peptide 2 at pH 7.09 withabout 0.5 equiv. NiCl₂. FIG. 13D is Peptide 2 at pH 7.20 with about 1equiv. NiCl₂.

FIGS. 14A-E are the ¹H-NMR spectra of Peptide 3(Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys, residues 1-12 of SEQ. ID.NO. 1). FIG. 14A is Peptide 3 at pH 2.83. FIG. 14B is Peptide 3 at pH7.15. FIG. 14C is Peptide 3 at pH 7.28 with about 0.13 equiv. NiCl₂.FIG. 14D is Peptide 3 at pH 7.80 with about 0.25 equiv. NiCl₂. FIG. 14Eis Peptide 3 at pH 8.30 with about 0.50 equiv. NiCl₂.

FIGS. 15A-D are the ¹H-NMR spectra of Peptide 4(His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys, residues 3-12 of SEQ. ID. NO.1). FIG. 15A is Peptide 4 at pH 2.72. FIG. 15B is Peptide 4 at pH 7.30.FIG. 15C is Peptide 4 at pH 8.30 with about 0.5 equiv. NiCl₂. FIG. 15Dis Peptide 4 at pH 8.10 with about 1 equiv. NiCl₂.

FIGS. 16A-D are the ¹H-NMR spectra of Peptide 5(Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys, residues 4-12 of SEQ. ID. NO. 1).FIG. 16A is Peptide 5 at pH 2.90. FIG. 16B is Peptide 5 at pH 7.19. FIG.16C is Peptide 5 at pH1. 02 with about 0.3 equiv. NiCl₂. FIG. 16D isPeptide 5 at pH 7.02 with about 0.6 equiv. NiCl₂.

FIGS. 17A-D are ¹H-NMR spectra of the N-terminal tetrapeptide,Asp-Ala-His-Lys, residues 1-4 of SEQ. ID. NO. 1. FIG. 17A is at pH 2.49.FIG. 17B is at pH 7.44. FIG. 17C is Peptide 3 at pH 7.42 with about 0.8equiv. NiCl₂. FIG. 17D is at pH 7.80 with about 1 equiv. NiCl₂.

FIGS. 18A-C are ¹H-NMR spectra of the N-terminal tetrapeptide withCoCl₂. FIG. 18A is at pH 7.44. FIG. 18B is at pH 7.23 with about 0.3equiv. CoCl₂. FIG. 18C is at pH 7.33 with about 0.8 equiv. CoCl₂.

FIGS. 19A-C are ¹H-NMR spectra of the N-terminal tetrapeptide withCuSO₄. FIG. 19A is at pH 7.31. FIG. 19B is at pH 7.26 with about 0.5equiv. CuSO₄. FIG. 19C is at pH 7.32 with about 1.0 equiv. CuSO₄.

DETAILED DESCRIPTION OF THE INVENTION

A number of terms used herein have the following definitions.

“Albumin-metal complex” or “metal-albumin complex” means the complex ofa divalent cation, including but not limited to copper, cobalt andnickel, to the N-terminus of naturally-occurring albumin.

“Albumin N-terminus” refers to that portion of naturally-occurringalbumin constituting comprising at least the four N-terminal aminoacids, i.e., Asp-Ala-His-Lys.

“Albumin N-terminal derivatives” refers to those species of albumin thatare altered or truncated at the N-terminus as a result of an ischemicevent. Specifically, the derivatives include those albumin specieslacking 4, 3, 2 and 1 N-terminal amino acid, as well as a full-lengthalbumin that is acetylated at its terminal Asp residue. Albumin-terminal derivatives cannot form albumin-metal complexes and may befound in the blood of ischemic patients. Full-length,naturally-occurring albumin is set forth is SEQ. ID. NO. 1.Acetylated-Asp albumin is set forth in SEQ. ID. NO. 2.

“Antibody to an albumin-metal complex” is an antibody to the epitopeformed of the metal and surrounding amino acids and/or their sidechains.

“Derivative N-terminus” refers to the 4-12 amino acids at the N-terminusof albumin N-terminal derivatives, which may serve as an epitope in thegeneration of a monoclonal antibody.

“Endogenous copper” refers to copper present in a patient sample ofalbumin, i.e., not exogenously added during the diagnostic procedure.

“Excess quantity” of metal ion or “excess metal ion” refers to additionof an amount of metal ion that will substantially exceed thestoichiometrically available albumin metal ion binding sites such thatsubstantially all naturally-occurring albumin is bound to metal ion atits N-terminus.

“Known value” as used herein means a clinically-derived cut-off value ora normal range, to which a measured patient value is compared so as todetermine the occurrence or non-occurrence of an ischemic event.

“Naturally-occurring albumin” refers to albumin with an intactN-terminus (Asp-Ala-His-Lys-) that has not been acetylated.

“Purified albumin” or “purified albumin sample” refers to albumin thathas been partially purified or purified to homogeneity. “Partiallypurified” means with increasing preference, at least 70%, 80%, 90% or95% pure.

“Treadmill test” means a stress test to increase myocardial O₂ demand,while observing if a mismatch occurs between demand and supply byobserving symptoms such as shortness of breath, chest pain, EKG, lowblood pressure and the like.

While not being bound by any particular theory, it is believed that thepresent method works by taking advantage of alterations which occur tothe albumin molecule, affecting the N-terminus of albumin during anischemic (“oxygen-depletion”) event. (Ischemia occurs when human tissueis deprived of oxygen due to insufficient blood flow.) A combination oftwo separate phenomena are believed to explain the mechanism by whichthe ischemia test of the present invention works. First, it is believedthat the localized acidosis which occurs during an ischemic eventgenerates free radicals which alter albumin's N-terminus; thus, bydetecting and quantifying the existence of altered albumin, ischemia canbe detected and quantified. Second, the acidotic environment presentduring ischemia results in the release of bound copper (fromceruloplasmin and other copper-containing proteins) which is immediatelytaken up by albumin. The bound copper also alters the N-terminus ofalbumin. (Not only does the presence of the complexed copper effectively“alter” the N-terminus, the metal ion damages the protein structure onbinding.) Thus, by detecting and quantifying the existence of alteredalbumin, and/or the copper-albumin complexes, ischemia can be detectedand quantified.

The details of the first mechanism are believed to be as follows. In theevent of an oxygen insufficiency, cells convert to anaerobic metabolism,which depletes ATP, resulting in localized acidosis and lowered pH, andcausing a breakdown in the energy cycle (ATP cycle). Cellular pumps thatkeep calcium against the gradient are fueled by energy from the ATPcycle. With ATP depletion, the pumps cease to function and cause aninflux of calcium into the cell. The excess intracellular calciumactivates calcium-dependent proteases (calpain, calmodulin), which inturn cleave segments of xanthine dehydrogenase, transforming thesegments into xanthine oxidase. The enzymes involved in this process aremembrane-bound and exposed to the outside of the cell, and are thus incontact with circulating blood. Xanthine oxidase generates superoxidefree radicals in the presence of hypoxanthine and oxygen. Superoxidedismutase dismutates the oxygen free radicals, turning them intohydrogen peroxide. In the presence of metals such as copper and ironwhich are found in blood, hydrogen peroxide causes hydroxyl freeradicals to be formed. Hydroxyl free radicals in turn cause damage tocells and human tissue. One of the substances damaged by free radicalsis the protein albumin, a circulating protein in human blood;specifically believed to be damaged is the N-terminus of albumin,resulting in the albumin N-terminal derivatives.

Human serum albumin is the most abundant protein in blood (40 g/l) andthe major protein produced by the liver. Many other body fluids alsocontain albumin. The main biological function of albumin is believed tobe regulation of the colloidal osmotic pressure of blood. The amino acidand structure of human albumin have been determined. Specifically, humanalbumin is a single polypeptide chain consisting of 585 amino acidsfolded into three homologous domains with one free sulfhydryl group onresidue #34. The specific amino acid content of human albumin is:

Residues: Asp Asn Thr Ser Glu Gln Pro Gly Ala Cys Val Met Ile Leu TyrPhe His Lys Trp Arg Number 39  15 30  22  60  23  25  12  63  35  39   6   8   61  18  30  16  58  1  23

In the first embodiment of the present invention, an excess of metal(e.g., cobalt) ions are introduced into a (purified) albumin sampleobtained from a patient serum, plasma, fluid or tissue sample (thisembodiment is hereafter referred to as the Aexcess metal embodiment”).In normal (non-ischemic) patients, cobalt will bind to one or more aminoacid chains on the N-terminus of albumin. In ischemic patients, however,most likely due to the alteration of the binding site of the N-terminus,cobalt binding to albumin is reduced. Accordingly, the occurrence ornon-occurrence of an ischemic state can be detected by the presence andquantity of bound or unbound cobalt. Measurement of cobalt can beconducted by atomic absorption, infrared spectroscopy, high-performanceliquid chromatography (“HPLC”) or other standard or non-standardmethods, including radioactive immunoassay techniques.

The details of the second mechanism are believed to be as follows.Ceruloplasmin is a circulating protein which binds copper; approximatelyninety-percent of the in vivo copper (copper is abundant in blood, withconcentrations comparable to iron) will be bound to ceruloplasmin. Theremainder is in other bound forms; almost no free copper exists incirculating blood. In acidic conditions and reduced oxygen conditions,such as happens during ischemia, ceruloplasmin releases some of itsbound copper. The released copper is taken up by albumin. Copper andcobalt both bind to albumin at the same site within the N-terminus.Thus, the bound endogenous copper, present during ischemia, blockscobalt from binding to albumin. The decrease in cobalt binding capacityof circulating albumin can be measured and quantified as a means fordetecting and quantifying the presence of an ischemic event.

The excess metal embodiment of the present invention comprises a methodfor detecting the occurrence or non-occurrence of an ischemic event in apatient comprising the steps of: (a) contacting a biological samplecontaining albumin of aid patient with an excess quantity of a metal ionsalt, said metal ion capable of binding to the N-terminus of naturallyoccurring human albumin, to form a mixture containing bound metal ionsand unbound metal ions, (b) determining the amount of bound metal ions,and (c) correlating the amount of bound metal ions to a known value todetermine the occurrence or non-occurrence of an ischemic event. In thismethod, said excess quantity of metal ion salt may comprise apredetermined quantity and the quantity of unbound metal ions may bedetected to determine the amount of bound metal ions. Additionally, thecompound selected from the group consisting of Asp-Ala-His-Lys-R,wherein R is any chemical group capable of being detected when bound toa metal capable of binding to the N-terminus of naturally occurringhuman albumin, may be utilized to facilitate detection.

This method uses samples of serum or plasma, or purified albumin.Preferred embodiments also include use of a metal ion salt comprising asalt of a transition metal ion of Groups 1b-7b or 8 of the PeriodicTable of the elements, a metal selected from the group consisting of V,As, Co, Cu Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and Ag. Alsopreferred, is detection of the amount of bound metal ions (or, in thecase where the excess quantity of metal ion salt is a predeterminedquantity, detection of the quantity of unbound metal ions) by atomicabsorption or atomic emission spectroscopy or immunological assay. Thesedetection mechanisms are also preferred for determination of thequantity of the compound Asp-Ala-His-Lys-R which is complexed with themetal ion salt in order to detect the quantity of unbound metal ions. Apreferred method for conducting said immunological assay is using anantibody specific to an antigen comprising the compoundAsp-Ala-His-Lys-R, wherein R is said metal ion.

Where the metal employed in the above excess metal embodiment is nickel,another preferred detection method is nuclear magnetic resonance (NMR).It has been observed that addition of Ni ion gives a sharp diamagnetic¹H-NMR spectrum for the resonances of the first three amino acids(Asp-Ala-His) of the albumin N-terminus octapeptide. While Co ion canalso induce changes in the NMR spectrum of the first three amino acidsof albumin, it induces paramagnetism at the binding site, resulting inbroadening of the resonances associated with the first three residues.Thus, the diamagnetic nature of the nickel complex makes it moreamenable for NMR studies.

The excess metal embodiment of the present invention also includes acolorimetric method of detecting the occurrence or non-occurrence of anischemic event in a patient comprising the steps of: (a) contacting abiological sample containing albumin of said patient with apredetermined excess quantity of a salt of a metal selected from thegroup consisting of V, As, Co, Cu, Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd,Fe, Pb, Au and Ag, to form a mixture containing bound metal ions andunbound metal ions, (b) contacting said mixture with an aqueous colorforming compound solution to form a colored solution, wherein saidcompound is capable of forming color when bound to said unbound metalion, (c) determining the color intensity of said colored solution todetect the presence of unbound metal ions to provide a measure of boundmetal ions, and (d) correlating the amount of bound metal ions to aknown value to determine the occurrence or non-occurrence of an ischemicevent. Preferred embodiments of this method include the additional stepof diluting said colored solution with an aqueous solution isosmoticwith blood serum or plasma prior to step (c). Also preferred are: usingferrozine as the color forming compound, and, alternatively, using thecompound Asp-Ala-His-Lys-R, wherein R is any group capable of formingcolor when bound to said metal ion as the aqueous color formingcompound. Conducting steps (b) and (c) in a pH range of 7 to 9 ispreferred. Further, conducting steps (b) and (c) using aspectrophotometer is preferred. Preferred samples in this method includeserum, plasma, or purified albumin and a preferred metal ion salt iscobalt.

Another embodiment is based on the endogenous copper mechanism discussedabove. This embodiment involves a method for detecting the occurrence ornon-occurrence of an ischemic state in a patient comprising the stepsof: (a) detecting the amount of endogenous copper ions present in apurified albumin sample of said patient, and (b) correlating thequantity of copper ions present with a known value to determine theoccurrence or non-occurrence of an ischemic event. Preferred methods fordetection of the amount of copper ions present in the purified albuminsample are by atomic absorption, atomic emission spectroscopy andimmunological assay. A preferred method of conducting said immunologicalassay uses an antibody specific to an antigen comprising the compoundAsp-Ala-His-Lys-R, wherein R is copper. This embodiment is referred toas the endogenous copper method.

Another embodiment of the subject invention is also based on the firstmechanism described above. The free radicals released during an ischemicevent damage the N-terminus of albumin by causing the cleavage of up tofour N-terminal amino acid residues, and possibly may induce acetylationof the N-terminus. The resulting albumin derivatives lack the capacityto bind to metal ions such as cobalt ion. In the subject embodiment, anischemic event is diagnosed by detecting the albumin derivatives thatcannot bind metal ion. For this reason, the subject embodiment isreferred to herein as the “derivative embodiment.”

As is reported in the Examples, albumin having an acetylated terminalAsp or lacking four, three, two or even one N-terminal amino acid havebeen found to lack the capacity to bind to cobalt ion. It has beenobserved that albumin derivatives lacking four, three, two or oneN-terminal amino acids are present in the serum or patients withischemia.

The derivative embodiment of the subject invention comprises a method ofdetecting or measuring an ischemic event in a patient by: (a) contactinga patient sample comprising naturally-occurring albumin and optionallyalbumin N-terminal derivatives with an excess quantity of metal ion thatbinds to the N-terminus of naturally-occurring albumin, wherebyalbumin-metal complexes are formed; (b) partitioning the complexes fromsaid derivatives, if any; (c) measuring at least one of saidderivatives, if any; and (d) comparing said measured derivative to aknown value, whereby the ischemic event may be detected or measured.

The derivative embodiment method can be practiced with a metal ion saltthat is a salt of a transition metal ion of Groups 1b-7b or 8 of thePeriodic Table of the Element. Preferably, the metal ion salt is a saltof a metal selected from the group consisting of V, As, Co, Sb, Cr, Mo,Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and Ag. Most preferred is that themetal ion is Ni or Co. The minimum incubation period for metal ion andalbumin is at least 4-5 minutes, and preferably 10 minutes, i.e., anamount of time sufficient for equilibrium to be reached. It is alsopreferred that heparin be added to the sample prior to the addition ofthe excess quantity of metal ion.

The partitioning step of the derivative embodiment method can be carriedout in two ways. It can be effected by having the excess metal ion ofstep (a) bound to a solid support such that the resulting albumin-metalcomplexes are retained on the solid support, permitting the elutionseparation of the albumin N-terminal derivatives. Alternatively, asolution of excess metal ion can be added to the patient sample,permitting the albumin-metal complexes to form, and the partitioning canbe effected by contacting the complexes with antibodies to themetal-albumin complex that are bound to a solid support.

Thus, in one aspect, the derivative embodiment involves a methodcomprising: (a) contacting a patient sample comprisingnaturally-occurring albumin and optionally albumin N-terminalderivatives with an excess quantity of a metal ion bound to a solidsupport, whereby the metal ion binds to the N-terminus ofnaturally-occurring albumin, forming metal-albumin complexes; (b)separating the complexes from said derivatives, if any; (c) measuring atleast one of said derivatives, if any; and (d) comparing said measuredderivative to a known value, whereby the ischemic event may be detectedor measured. It is preferred that the solid support of step (a) be adiacetate or a phosphonate matrix. It is also preferred that the metalion used in step (a) be nickel ion. It is further preferred that copperion not be used in this method as it is likely to demonstratenon-specific binding to albumin thiol groups (located outside theN-terminus), possibly generating false negative results.

Metal affinity chromatography methods useful in this embodiment arewithin the skill in the art. For example, resins for separating proteins(including albumin) using metal affinity chromatography are described inU.S. Pat. Nos. 4,569,794; 5,169,936; and 5,656,729.

In another aspect, the derivative embodiment involves a methodcomprising: (a) contacting a patient sample comprisingnaturally-occurring albumin and optionally albumin N-terminalderivatives with an excess of a metal salt, whereby a metal-albumincomplex is formed; (b) contacting the mixture of step (a) with anantibody to said complex, said antibody being bound to a solid support;(c) separating the complex from said N-terminal derivatives, if any; (d)measuring the amount of at least one N-terminal derivative, if any; and(e) comparing the measured N-terminal derivative to a known value,whereby all ischemic event may be detected or measured. In this aspect,it is preferred that the metal ion be cobalt ion.

The step of measuring the albumin N-terminal derivatives can be carriedout using antibodies (monoclonal or polyclonal) to the derivatives. Theantibodies can be directed to one or more of the N-terminal epitopes foreach derivative. Thus, one or more antibodies directed to one or moreN-terminal epitopes may be used to measure the derivatives.Additionally, measuring can be accomplished by employing anantibody(ies) to albumin non-N-terminal epitopes. Because thepartitioning step has removed all naturally-occurring albumin, anyremaining albumin will be an N-terminal derivative. Antibodies used inthe measuring step are labeled, preferably with an enzyme or afluorescent label or by other methods known in the art.

The derivative embodiment methods can be carried out using kits havingcomponents adapted to provide the reactants or reagents and carry outthe process steps. Where the derivative embodiment method involvesexcess metal ion bound to a solid support, the kit illustrated in FIG. 1can be employed. Referring to FIG. 1, the diagnostic kit 20 isconstructed of an upper plate 1 and lower plate 3. The lower plate 3 has1-2 elongated solid supports 6 (e.g., nitrocellulose) with a sampleapplication filter 4 upon which a patient sample is applied throughsample port 2. The filter 4 and port 2 may be positioned such that thefilter 4 is common or shared by both elongated solid supports 6. Thefilter 4 removes cells (red and white blood cells, platelets, etc.),permitting plasma to flow through to supports 6. The patient samplemigrates from the filter at the first end of each of the elongated solidsupports 6 to the second ends at the end of process indicators 18. Thefirst solid support 6 provides a test function and the second provides acontrol function. The solid support providing a test function has anarea 8 of immobilized metal ion to which naturally-occurring albuminbinds. The albumin N-terminal derivatives continue to migrate down thesolid support 6 to an area 10 containing ligand. In preferredembodiments, the ligands at area 10 are antibodies to albumin N-terminalderivatives and/or antibodies to naturally-occurring albumin. Anantibody to naturally-occurring albumin may be used at area 10 providedit is directed to an epitope that is not located at the N-terminus ofnaturally-occurring albumin, so that it may bind to the derivatives. Anantibody at area 10 to an albumin N-terminal derivative refers to anantibody directed to an N-terminal epitope of the derivative, such thatthe antibody is specific (i.e., recognizes only) the particular albuminN-terminal derivative. An advantage of including antibodies to albuminN-terminal derivatives at area 10 is that the amount of each or allN-terminal derivatives can be measured. Measurement of each derivativemay permit a more accurate assessment of the degree and timing of theischemic event. For example, a relatively higher concentration of thederivative lacking four N-terminal amino acids may reflect a greaterdegree or a longer duration of ischemia than a second sample whereanother derivative (e.g., albumin lacking only its N-terminal Aspresidue) is more prevalent. Although the relative order of appearance ofeach derivative during the course of an ischemic event has not yet beendetermined, it will be possible to do so upon correlation of derivativesobserved in patient samples with clinical observations of patients fromwhom the samples have been derived.

In the control (second) elongated solid support 6, an area 11 containingligand to albumin is provided to detect all albumin, naturally-occurringor N-terminal derivatives, in the sample. Thus, the antibody at area 11is directed to an albumin epitope that is not located at the N-terminusof albumin. The antibody or antibody mixture at areas 10 and 11 shouldbe the same for control purposes.

The test and control results can be observed through ports 12 and 14,respectively. The binding of albumin or albumin N-terminal derivativesto antibody is detected by methods known in the art such as sandwichassays, enzyme assays or color indicators. For example, a labeledantibody may be added through ports 12 and 14 to bind to any albuminthat is bound to antibody attached to areas 10 and 11. The label on theadded antibody may be, for example, alkaline phosphatase, a commonlyused reporter enzyme which reacts with synthetic substrates such as1,2-doxetane or p-nitrophenylphosphate to yield detectable products.Alternatively, a protein coloring reagent such as bromo cresol purple orbromo cresol green may be present in areas 10 and 11 or added throughports 12 and 14.

Finally, an end of process indicator 18 at the second end of eachelongated solid support 6 may be employed to assure completion of thetest, i.e., that a sufficient volume of biological sample has passeddown each elongated solid support 6 for the test to be completed.Suitable end of process indicators 18 include pH indicators andconductance indicators as is known in the art.

The kit illustrated in FIG. 1 can also be used where the derivativeembodiment method employs a solid-support bound antibody to thealbumin-metal complex. Referring again to FIG. 1, the patient sample isfirst mixed with excess metal ion aqueous solution, wherebynaturally-occurring albumin-metal complexes are formed, and then appliedto the filter 4 at the first end of the elongated solid supports 6. Asthe sample migrates down the test (first) elongated solid support 6, itencounters area 8 between the first and second ends which hasimmobilized antibody to the albumin-metal complex. The albumin-metalcomplex binds to area 8, and the N-terminal derivatives continuemigration to area 10 containing ligand to albumin which is proximate thesecond end. The ligand at area 10 can be an antibody directed to analbumin epitope that is not located at the naturally-occurringN-terminus, or can be antibodies to derivative N-terminal epitopes. Anend of process indicator 18 can also be present at the second end of thefirst elongated solid support. A second or control elongated solidsupport 6 can also be present in the kit 20 with an area 11 havingimmobilized antibody to the albumin located between the first and secondends.

The subject invention provides additional kit embodiments suitable forthe derivative embodiment method employing the solid support boundantibody to albumin-metal complex. Referring now to FIG. 2, a kit 40 isprovided containing a solid support disk or circle 28 having a centrallylocated sample application filter 30 for application of a patient samplethat has been mixed with excess metal ion, whereby naturally-occurringalbumin-metal complexes have been formed. The circular filter issurrounded by an inner concentric ring divided into a test half 32 whichcontains ligand (e.g., monoclonal antibody) to albumin-metal complexes,and a control half 34 which contain no ligand. Beyond the innerconcentric ring is an outer concentric ring divided into a test half 38and a control half 36, both of which contain ligand to albumin. In area36, ligand is provided that detects all albumin, naturally-occurring orN-terminal derivatives, in the sample. Thus, the antibody at area 36 isdirected to an albumin epitope that is not located at the N-terminus ofnaturally-occurring albumin. In area 38, ligand to naturally-occurringalbumin and/or to albumin N-terminal derivatives is likewise provided.Again, for control purposes, the antibody or antibody mixture in areas36 and 38 should be the same.

As the patient sample radiates from the filter 30, the albumin-metalcomplexes bind to antibody to complexes in area 32. Filtrate from area32 passes into area 38, where albumin N-terminal derivatives bind toantibody. Likewise, as patient sample radiates through area 34 of thecontrol half and into area 38, all albumin present (naturally-occurringand derivative) binds to antibody present in area 36. The amount ofalbumin or albumin derivatives bound in area 38 is compared to a knownvalue to determine whether an ischemic event has occurred. The amount ofalbumin or derivatives in area 38 can also be compared to a scale ofknown values, such as a color scale, to determine the degree of theischemic event. The amount of albumin or derivatives bound in area 38 isdetermined by methods known in the art including sandwich assays, enzymeassays or protein color reagents.

As can be appreciated by those skilled in the art, the embodiment inFIG. 2 can also be readily adapted to the derivative embodiment methodin which metal ion is bound to the solid support. Specifically, thesolid support area 32 would have metal ion bound thereto rather thanantibody to albumin-metal complex.

FIG. 3 illustrates another kit 60 suitable for the derivative embodimentmethod employing the solid support bound antibody to albumin-metalcomplex. The kit 60 comprises a circular solid support 56 with acentrally located sample application filter 50. The filter 50 issurrounded by a concentric ring which is divided into two semi-circles.The control semi-circle contains an area 54 containing ligand tonaturally-occurring albumin and albumin derivatives, preferably anantibody directed to an albumin epitope not located at the N-terminus ofnaturally-occurring albumin. The test semi-circle contains an area 52containing ligand to albumin-metal complex. Thus, after a patient sampleis mixed with an excess metal ion solution, whereby albumin-metalcomplexes are formed, it is applied to filter 50 from which it radiatesto area 52, where the albumin-metal complexes bind to the ligand. In thecontrol semi-circle, the patient sample radiates and thenaturally-occurring albumin (complexes) and derivatives bind to theligand in area 54. The ligand in area 54 is preferably a monoclonal orpolyclonal antibody directed to a non-N-terminal epitope ofnaturally-occurring albumin. By comparing the amount of total albuminand derivatives bound to area 54 to the amount of albumin-metalcomplexes bound to area 52, the amount of albumin derivatives can becalculated or estimated, and an ischemic event detected or measured. Thealbumin or derivatives bound to antibodies on each area (52 or 54) canbe detected or measured by methods known in the art including sandwichassays, enzyme assays and protein color assays.

FIG. 3 can likewise be adapted to be useful in the derivative embodimentmethod in which metal ion is bound to the solid support, i.e., wheremetal ion is immobilized in area 52.

As is discussed above, a variety of antibodies are employed in thevarious embodiments of the subject invention. In the excess metal,endogenous copper and derivative embodiments, antibodies toalbumin-metal complexes are employed. Patient antibodies specific to thealbumin-metal (cobalt and nickel) complexes (including the N-terminalepitope) have been identified in occupational studies (Nieboer et al.(1984) Br. J. Ind. Med. 41:56-63; Shirakawa et al. (1992) Clin. Exp.Allergy 22:213-218; Shirakawa et al. (1990) Thorax 45:267-271; Shirakawaet al. (1988) Clin. Allergy 18:451-460; and Dolovich et al. (1984) Br.J. Ind. Med. 41:51-55). Additionally, rabbit antibodies to humanalbumin-metal complexes have also been generated (Veien et al. (1979)Contact Dermatitis 5:378-382). Therefore, antibodies to albumin-metalcomplexes for use in the subject methods either already exist in the artor would be readily obtainable using known methods.

In addition to the foregoing antibodies, the derivative embodiment mayalso use antibodies to one or more of the albumin N-terminalderivatives. As is set forth in the Examples, it has been found that thealbumin derivatives that lack four, three, two and even one N-terminalamino acid have lost the capacity to bind to cobalt. Additionally,full-length albumin that has been acetylated at its Asp residue alsocannot bind to cobalt. As is appreciated by the skilled artisan,antibodies that are specific to (i.e., recognize only) each of thesederivatives can be obtained using known monoclonal antibody technology.Adjuvants such as KLH may be used to enhance immunogenicity.

Applications, embodiments and methods of the present inventioncomprising one or more of the aforementioned methods of the presentinvention include: A method for ruling-out the existence of ischemia ina patient, comprising application of any of the aforementioned methods,including application of any of the subject methods wherein said patientpossesses one or more cardiac risk factors, said cardiac risk factorsbeing selected from the group consisting of: age greater than 50,history of smoking, diabetes mellitus, obesity, high blood pressure,high cholesterol, and strong family history of cardiac disease. Avariant thereof, comprises subjecting the patient to an exercisetreadmill test followed by a second application of the same method,followed by a comparison of the results of the two applications.Comparison of the before and after ischemia diagnostic tests will revealwhether the ischemic event is induced only under the elevated metabolicconditions of exercise. This method may be used to detect the existenceof ischemia provoked by exercise in an otherwise asymptomatic patient.

Other embodiments, applications and variants of the present inventioninclude a method for ruling-out the occurrence of an temporally-limitedischemic event in a patient comprising application of any of the subjectdiagnostic methods; a method of detecting the existence of ischemia inan asymptomatic patient comprising application of any of the subjectdiagnostic methods; a method for the evaluation of patients sufferingfrom stroke-like signs to determine the occurrence or non-occurrence ofa stroke, comprising application of any of the subject diagnosticmethods; a method for distinguishing between the occurrence of anischemic stroke and a hemorrhagic stroke, comprising application of anyof the subject diagnostic methods; and a method for assessing theefficacy of an angioplasty procedure, comprising application of any ofthe subject diagnostic methods.

The present invention also provides a method for evaluation of a patientpresenting with angina or angina-like symptoms to detect the occurrenceor non-occurrence of a myocardial infarction, comprising application ofany of the subject diagnostic methods and application of anelectrocardiographic test, followed by correlation of the results of theapplication of the diagnostic method with the results of theelectrocardiographic test to determine the occurrence or non-occurrenceof a myocardial infarction. Preferred electrocardiographic tests areE.C.G., E.K.G. and S.A.E.C.G. tests.

Another method of the present invention is a method for supplementingelectrocardiographic results to determine the occurrence ornon-occurrence of an ischemic event, comprising application of any ofthe subject diagnostic methods and application of anelectrocardiographic test, followed by correlation of the results ofapplication of the diagnostic method with the results of saidelectrocardiographic test to determine the occurrence or non-occurrenceof an ischemic event. A variant thereof, comprises application of themethod wherein said patient is undergoing surgery.

A further method of the present invention is a method for comparinglevels of ischemia in patients at rest and during exercise is alsotaught by the present invention, comprising application of the followingsteps at designated times: (a) application of any of the subjectdiagnostic methods at a first designated time, (b) administration of anexercise treadmill test followed by a second application of the samediagnostic method employed in step (a), (c) comparing the results of theapplication of the diagnostic method prior to administration of theexercise treadmill test with the results of the application of thediagnostic method after administration of the exercise treadmill test,and (d) repeating steps (a) through (c) at additional designated timeswherein, results obtained at designated time are compared. Thisembodiment may be used to evaluate patients with known or suspectedischemic conditions, to assess the patency of an in-situ coronary stentand to assess the efficacy of an angioplasty procedure. Preferreddesignated time intervals are three months, six months or one year.

The present invention also teaches a method for assessing the efficacyof thrombolytic drug therapy, comprising the application of any of thesubject diagnostic methods; and a method for detecting in a pregnantwoman the occurrence of placental insufficiency, comprising applicationof any of the subject diagnostic methods.

The subject invention also includes calibration standards which haveknown molar ratios of albumin and metal and are useful in calibratinganalyzers or kits that employ the subject methods. In one embodiment,the calibrator compositions are standards to be used to generatestandard curves for calibration of clinical chemistry analyzers such asthe Beckman CX-5™, Roche Cobas Mira™ and Dimension XL™. These analyzerscan each detect or measure ischemic events based on the calorimetricversion of the excess metal embodiment described herein. The calibratorcompositions can also be used to calibrate analyzers such as atomicabsorbance or atomic emission spectrophotometers. The calibratorcompositions have preselected or predetermined ratios ofnaturally-occurring albumin and metal ion. In preferred embodiments, thealbumin is human, the solution is buffered (e.g., Tris or HEPES), the pHis about 7-8, and the metal is divalent and is selected from the groupconsisting of cobalt, nickel and copper. Aliquots of these calibrators,under specific conditions, produce a defined absorbance at 470-500 nm,i.e., a standard curve.

The albumin that is used in the calibrators is substantially allnaturally-occurring. By “substantially all,” it is meant that at least70%, and with increasing preference, at least 80%, 90% and 95% byweight, of the albumin is naturally-occurring. Without wishing to bebound by theory, it is believed that when the calibrator compositionsare placed in solution, the metal ion becomes primarily bound to theN-terminus of the albumin, although it is possible that a minor amountof metal ion can be bound to thiol or other groups located on thealbumin.

The calibrators are typically manufactured by starting with initialconcentrated solutions of albumin and metal salt, and then mixing theseconcentrates in defined ratios to obtain desired molar ratios of albuminand metal concentrations in the resulting calibrator solutions.

To generate the standard curve for the colorimetry-type analyzers, eachof the calibrator solutions is mixed with a known, constant amount ofexcess metal salt and excess coloring reagent as described herein.Thereafter, absorbance is measured at 500 nm and blocked albumin isplotted against absorbance. Because the amount of metal originallypresent in the calibrator solution and the excess metal salt added areboth known, the absorbance, which is associated with the excess metalion that did not bind to albumin, can be correlated with degree ofN-terminal blockage of albumin originally present in the calibratorsolution. As the degree of N-terminal blockage, i.e., percentage oforiginal metal concentration, in the calibrator solution increases, theabsorbance due to excess metal ion that does not bind to albumin alsoincreases. The relationship is linear.

To generate the standard curve for the atomic absorbance or atomicemission spectrophotometer, the calibrator solutions are applied to theanalyzer. The absorbance is plotted against the original metalconcentration present in each calibrator to generate the standard curve.

Thus, the calibrator solutions are designed and intended to mimicischemic patient samples in reflecting a range of albumin that isalready bound to metal ion and is unavailable for binding to exogenouslyadded metal ion. For example, a calibrator solution that has 75% of itsalbumin blocked with Cu at its N-terminus has only 25% of its albuminavailable for binding to exogenous, excess Co. After addition ofcoloring reagent to react with unreacted Co, absorbance at 500 nm willbe much greater than that which would be observed for a calibratorsolution that is only 25% blocked with Cu at its N-terminus.

For quality control purposes, the characteristics of the calibrators canbe verified by:

1. measuring their metal to albumin ratio; metal can be measured byatomic absorption, and albumin can be measured by bromo cresol green(BCG) assay;

2. using radioactive Co⁵⁷ albumin binding assay employing a Sepharosecolumn;

3. measuring the absorbance of the calibrators at the appropriatewavelength over time; and

4. measuring the absorbance of mixtures of calibrator solutions andexcess cobalt plus coloring reagent, such as dithiothreitol (DTT).

A greater understanding of the present invention and of its manyadvantages may be had from the following examples, given by way ofillustration. The following examples are illustrative of some of themethods, applications, embodiments and variants of the presentinvention. They are, of course, not to be considered in any waylimitative of the invention. Numerous changes and modification can bemade with respect to the invention.

EXAMPLE 1 Sample Handling Procedures for Ischemia Testing

The samples which were used in the present invention were obtained froma variety of tissues or fluid samples taken from a patient, or fromcommercial vendor sources. Appropriate fluid samples included wholeblood, venous blood, arterial blood, blood serum, plasma, as well asother body fluids such as amniotic fluid, lymph, cerebrospinal fluid,saliva, etc. The samples were obtained by well known conventional biopsyand fluid sampling techniques. Preferred samples were blood plasma andserum and purified albumin. Purified albumin was isolated from the serumby any of the known techniques, including electrophoresis, ion exchange,affinity chromatography, gel filtration, etc.

Blood samples were taken using Universal Precautions. Peripheralvenipuncture was performed with the tourniquet on less than 30 seconds(contralateral arm from any IV fluids). Blood is drawn directly into two10 cc Becton Dickinson Vacutainer® Sodium-Heparinized tubes and wasgently inverted once to mix. If an IV port was in use, the blood wascollected (after a discard sample was drawn equivalent to the dead spaceof usually 5 cc) into a plain syringe and dripped gently down the sideof two 10 cc Becton Dickinson Vacutainer® brand tubes and gentlyinverted once to mix. Blood was also collected directly from theVacutainer® tubes with special administration sets with a reservoirsystem that does not require a discard sample. These systems allow adraw to be taken proximal to the reservoir.

Plasma tubes were centrifuged within 2 hours of the draw. (Note,collected serum was clotted between 30-120 minutes at room temperature(RT) before centrifugation. The inside of the serum tube was ringed witha wooden applicator to release the clot from the glass beforecentrifugation. If the subject was taking anti-coagulants or had a bloodclotting dysfunction, the sample was allowed to clot longer than 60minutes, between 90-120 minutes was best.) The tubes were centrifugedfor 10 minutes at RT at 1100 g (≦1300 g). Collected samples were pooledin a plastic conical tube and inverted once to mix.

If the sample was not used within 4 hours of centrifugation, the samplewas frozen. Alternatively, separated serum was refrigerated at 4° C.until tested, but was tested within 8 hours (storage over 24 hours mayhave resulted in degradation of the sample). “Stat” results (obtainedwithin 1 hour of completion of centrifugation step) were preferred. Thefollowing percent differences for the ischemia test were measured usingplasma and serum samples ≦8 hours and ≦24 hours after collection.Delayed test results were compared to stat test results on the samepatient sample and the mean percent differences (and standarddeviations) were as given below:

Storage and Delayed Testing Data for the Ischemia Test ≦8 hrs. vs. stat≦24 hr. vs. stat* Plasma n 20 n 23 (stored at % diff   −5.3% % diff  −4.8% room temp) S.D.    .094 S.D.    .090 Plasma n 18 n 40 (stored at% diff     1.7% % diff     1.0% 4° C.) S.D.    .070 S.D.    .094 Serum n16 (stored at % diff   −12.8% (not enough room temp) S.D.    .157samples) Serum n 14 n 24 (stored at % diff   −7.3% % diff   −2.7% 4° C.)S.D.    .040 S.D.    .210 *≦24 hr. test results given here are a totalthat include the ≦8 hr. test sample results.

EXAMPLE 2 Test Method for Detecting Occurrence of Ischemic Event UsingCobalt Binding

The ischemia test (cobalt version) was run as follows: 200 μl of patientsera was added to each of two tubes each containing 50 μl 0.1%CoCl₂.6H₂O. The mixture was allowed to react at room temperature (18-25°C.), or higher, for 5 or more minutes. Thereafter 50 μl 0.01 Mdithiothreitol (DTT) was added to one of the two tubes (the “test tube”)and 50 μl 0.9% NaCl was added to the second tube (the “backgroundtube”). After two minutes, 1 ml 0.9% NaCl was added to both tubes. A470spectroscopy measurements were taken of the two tubes. The ischemia testwas considered positive if the optical density was greater than or equalto 0.400 OD (or alternatively a clinically derived cut-off) using aspectrophotometer at OD 470 nm.

Equivalent materials which may be used as alternatives include any ofthe transition metals. Ferrozine or other compounds with an affinity tocobalt can be substituted for DTT and/or any cobalt or metal coloringreagent. CoCl₂.6H₂O, for instance, can be utilized. The optimal rangefor cobalt binding to albumin is from pH 7 to pH 9, with a range of pH7.4-8.9 being most preferred; pH 9 is optimal for cobalt interactionwith the color reagent. The amount of serum sample can also vary, as canthe amounts of CoCl₂.6H₂O and DTT and ferrozine. Critical, however, isthat the amount of cobalt used be in excess of the amount of albumin andthat the DTT or ferrozine be in excess of the cobalt.

EXAMPLE 3 Test Method for Detecting Occurrence of Ischemic Event UsingMeasurement of Copper

Albumin was purified from 0.2 cc of human serum or plasma using an ionexchange method to produce approximately 8 mg of purified albumin. Abuffer having a pH in the range of 7 to 9 was added. The amount ofcopper present in the sample was then measured by directspectrophotometric and potentiometric methods, or by any of severalother known methods, including atomic absorption, infrared spectroscopy,HPLC and other standard or non-standard methods, including radioactivetracer techniques. The proportion of copper to albumin can be then usedas a measure of ischemia, the greater the proportion, the higher theischemia value.

EXAMPLE 4 Test Method for Ruling-out the Existence of Ischemia in aPatient

The following protocol is designed to rule out ischemic conditions inhealthy appearing patients who describe prior symptoms of occasionalchest pain or shortness of breath.

First, a medical history (including a detailed history of the presentand past medical problems and risk factors for ischemic heart disease),physical exam, and vital signs are obtained. If the patient has anycardiac risk factor for ischemic heart disease (age >50, smoking,diabetes mellitus, obesity, high blood pressure, elevated low densitylipoproteins, high cholesterol, and strong family history of cardiacdisease), the physician is instructed to order a resting twelve-lead EKGand a chest x-ray. If the twelve-lead EKG shows evidence of an acutemyocardial infarction (AMI), the patient is immediately transported to ahospital for intensive cardiac treatment. If the twelve-lead EKG doesnot show evidence of (AMI), the patient will be scheduled for anoutpatient twelve-lead EKG exercise treadmill within the next few days.A blood sample should be drawn immediately before and again after theexercise treadmill test and the ischemia test run on each sample.

If the exercise treadmill test shows definite evidence of cardiacischemia, usually seen by characteristic changes of the ST segments,dramatic abnormalities of pulse or blood pressure, or anginal chestpain, the patient should be treated for cardiac ischemia and referred toa cardiologist for possible coronary angiogram and angioplasty. If theexercise treadmill test does not show any evidence of cardiac ischemia,or die findings are equivocal, but the ischemia test is abnormal, thepatient similarly should be treated for cardiac ischemia and referred toa cardiologist for possible coronary angiogram and angioplasty. (Absentthe present invention, such patients with moderate to high cardiac riskfactors would be referred to a cardiologist for further (typicallyinvasive) cardiac testing).

If the exercise treadmill test does not show any evidence of ischemicheart disease, or the findings are equivocal, and the isehemia test isnormal, the patient may be sent home with no evidence of cardiacisehemia. In comparison, prior to the present invention, in the casewhere the exercise treadmill test does not show any evidence of cardiacischemia, or the findings are equivocal, patients with low risk forcardiac isehemia typically would not have any other tests ordered. Insuch cases, the physician is taking a calculated risk. It is welldocumented in the medical literature that at least 25 to 55 percent ofpatients (higher in females) will have some isehemic heart disease whichis not found with routine exercise treadmill testing.

EXAMPLE 5 Test Method for Evaluating Patients with Angina to Rule-outthe Occurrence of an Ischemic Event

In this study, clinical criteria (EKG changes, elevated cardiac enzymesor markers, positive thallium treadmill or positive angiogram) were usedto determine the presence or absence of isehemia in patients presentingwith chest pain. Isehemic patients were those with at least one clinicalfinding positive for isehemia. Normal patients were those for whomclinical findings were negative, as well as normal volunteers with nohistory or symptoms of cardiac or cerebral isehemia.

Blood samples were taken from 139 subjects who either presented toemergency departments of several hospitals with chest pain or normalvolunteers. Blood was drawn into plain red top tubes and, after tenminutes, the clotted blood was centrifuged to separate the serum. Serumwas refrigerated at 4° C. until tested. If the sample would not be usedwithin 4 hours of centrifugation, it was frozen, but in no case wastesting delayed more than 3 days.

Samples were centrifuged for 5-10 minutes in an analytical centrifugeimmediately before testing. 200 μl off each sample was aliquoted intriplicate with an additional tube to be used as a Blank (no DTT)control into borosilicate glass tubes. Also aliquoted was 200 μl of aStandard, such as Accutrol or HSA, in triplicate plus a Blank control.At 10 second intervals, 50.0 μl of 0.10% CoCl₂ (store working stock andstock at 4° C.) was added to each tube. Solution was added to thesample, not glass, and tubes were “flicked” to mix.

After 10.0 minutes (starting with the first tube to which cobaltsolution was added) an additional 50.0 μl of 0.9% NaCl was added to thetwo Blank tubes using the appropriate 10 second intervals. 50.0 μl of0.01 M DTT was additionally added to the Plasma (not Blank) tubes intheir appropriate 10 second intervals. Of note, it is preferred that DTTbe made fresh weekly (6 mg per 4 ml H₂O) and stored at 4° C.

After 2 minutes (starting with the first tube to which cobalt solutionwas added) 1.0 ml of 0.9% NaCl solution was added to each tube, usingthe appropriate 10 second intervals. Tubes were agitated to mix. In theevent that there were too many tubes to finish the test tubes in 10second intervals, reagents were added to the “Blank” tubes withouttiming.

The optical density of each sample set was read using the set's Blank toread absorbance at 470 nm. The cuvette was checked for air bubblesbefore reading and washed with H₂O between sets. The ischemia test wasconsidered positive if the optical density was greater than or equal to0.400 using the spectrophotometer at OD 470 nm.

The results of the ischemia test compared to the diagnosis determined byclinical criteria are as described in the chart below. Four falsenegatives and three false positives were reported.

Ischemia Test Clinical Diagnosis + − + 99 95 4 − 40 3 37

Study results demonstrated that the ischemia test marker has a highervalue in patients with clinically diagnosed ischemia. The diagnosticaccuracy of the ischemia test for the chest pain study was above 90percent (sensitivity, 96.0%; specificity, 92.5%; predictive value,(+)96.9%; predictive value, (−) 90.2%).

EXAMPLE 6 Test Method for Evaluation of Patients Suffering from ChestPain to Determine the Occurrence or Non-occurrence of a MyocardialInfarction

The following study is proposed to test the ability of the presentinvention to detect ischemia in the initial hours following the onset ofchest discomfort suspicious for cardiac ischemia. The cobalt version ofthe test is used.

The patient population is limited to male or female persons, 30 years orolder, who present to the Emergency Department with complaints of chestdiscomfort of less than four hours in duration for reasons independentof the study. Patients will be excluded from the study if they meet anyof the following criteria: (1) known concurrent non-cardiac ischemicdisease(s), including but not limited to transient ischemic attacks,cerebral vascular accident, peripheral vascular disease, intermittentclaudication, bowel ischemia, and severe renal failure; (2) definiteradiological evidence of a cause of chest discomfort that is other thancardiac ischemia, such as, but not limited to, pneumonia, pneumothorax,and pulmonary embolus; or (3) chest discomfort temporally related tolocal trauma.

All standard evaluation and treatment appropriate for emergencydepartment patients with suspected cardiac ischemia will be followed atall times. The drawing of blood for the study will not in any mannermodify the standard treatment protocol. Within these parameters, apre-treatment evaluation will be conducted, which will includedocumentation of all current medications, documentation of previousmedical history, EKG, laboratory and radiographic test results, anddocumentation of most recent vital, signs and a physical examination.

The study consists of drawing an extra blood sample at the time ofadmission to the emergency department. Samples are collected from acatheter that is already in place for intravenous access oralternatively by venipuncture. Collection and administration of theischemia test is as described in Example 5 herein.

EXAMPLE 7 Test Method for Detection of Ischemia in Patient at Rest andDuring Exercise

The primary objective of this trial was to employ and test thesensitivity of the ischemia test at various time points, before, duringand after an exercise thallium treadmill test. Preliminary data hasshown that the blood level of the ischemia test (i.e., absorbance,cobalt excess metal embodiment) rises immediately after an ischemicevent. The purpose of this pilot investigation is to determine themagnitude of this rise in level of the ischemia test during a test todefine the presence or absence of a cardiac ischemic event, said testbeing the exercise thallium treadmill test. While it is possible thatpatients scheduled for exercise thallium treadmill test may have alreadyexperienced an ischemic event, preliminary data indicates that afurther, significant decline in cobalt binding (and an increase in theserum absorbance or unbound metal ion) will occur if tissue ischemia isinduced during the exercise thallium treadmill test.

Patients already scheduled for an exercise thallium treadmill test wereasked to give their consent for participation which required two tubesof blood (20 cc's) to be drawn up to 5 (five) times before, during andafter the exercise thallium treadmill test. Eligible patients consistedof patients who met all of the following criteria: (1) Age: 18 years orolder; (2) Male or female; (3) able to provide written informed consent;and (4) referred for exercise thallium treadmill test for reasonsindependent of this investigation. Patients were excluded fromparticipation in the study if they met any of the following criteria:(1) known concurrent non-cardiac ischemic disease including, but notlimited to: transient ischemic attacks, cerebral vascular accident,acute myocardial infarction and intermittent claudication; (2) inabilityto complete the standard protocol for the exercise portion of theexercise thallium treadmill test; or (3) cardiac arrest during theexercise portion of the exercise thallium treadmill test.

Prior to administration of the exercise thallium treadmill test, apretreatment evaluation was conducted which included documentation ofall current medications, documentation of previous medical history, EKG,laboratory and radiographic test results, and documentation of mostrecent vital signs and physical examination.

The standard exercise thallium treadmill test procedure was followed atall times. In no instance was the drawing of the additional bloodsamples for the purpose of the study permitted to subject the patient toadditional risk (beyond the drawing of blood), or to in any mannermodify the treatment of the patient.

The “standard” exercise thallium treadmill test procedure comprisedgenerally the following: The patient was brought to the exercise testroom in a recently fasting state. After initial vital signs and recenthistory was recorded, the patient was connected to a twelve-lead EKGmonitor, an intravenous line was established and the patient wasinstructed in the use of a treadmill. With the cardiologist inattendance, the patient walked on the treadmill according to thestandard Bruce protocol: starting at a slow pace (approx. 1.7 mph) andgradually increasing both the percent grade (slope) of the treadmill andthe walking speed at three minute intervals up to a maximum of 5.5 mphat 20° grade. Termination of the exercise portion on the exercisethallium treadmill test occurred at the discretion of the cardiologistbased on patient symptoms, EKG abnormalities, or the attainment of 85%maximal heart rate. With the patient near maximal effort on thetreadmill, approximately 3 mCi of thallium²⁰¹ was injected intravenouslywhile the patient continued to exercise for approximately one moreminute. At the end of exercise, single photon emission computerizedtomography (SPECT) was used to scan the patient's myocardium for anyperfusion defects. Following recovery, between 2 and 4 hours afterexercise, a smaller amount of thallium²⁰¹ (approximately 1.5 mCi) wasre-injected for repeat SPECT scan. EKGs and SPECT scans were analyzedfor ischemic criteria. The SPECT scans may show fixed and reversibleperfusion defects. The reversible perfusion defects indicate ischemiaand the fixed defects indicate myocardial scarring.

The study consisted of drawing blood samples on 3 occasions during theexercise thallium treadmill procedure. Two tubes of blood (approximately4 teaspoons) were collected before the exercise test, immediately afterexercise, and between 1 and 4 hours after exercise. Blood samples werecollected from the catheter already in place for the exercise thalliumtreadmill procedure or alternatively by venipuncture. Note: RadiationProtection/Safety Considerations—Blood drawn following thallium²⁰¹injection was routinely considered safe because the amount injected wasapproximately 3 mCi and, for all practical purposes, the dilution intothe systemic circulation reduces the sample level to less than 0.67nanoCi per cc.

Standard patient follow-up was conducted according to clinical practice.Patients who had subsequent coronary angiograms after being enrolled inthis exercise thallium treadmill test study had all resultant coronaryangiogram information obtained recorded to verify the exercise thalliumtreadmill test results.

All clinical and research laboratory testing procedures were performedin a blinded fashion.

Of the 59 patients enrolled (plasma and serum samples tested by theischemia test method), 11 patients were deleted because of one of thefollowing reasons: a chronically occluded coronary artery and no samplecollected later than one hour after exercise, a clinical history ofexercise leg pain (claudication), hemolyzed baseline blood samples,patient did not continue with the exercise study or did not agree tofurther blood tests, patient received an exercycle thallium test insteadof a treadmill thallium test and one patient whose chest pain was laterdetermined to be due to pneumonia.

Of the remaining 48 patients, 23 had no history of known ischemic heartdisease, 23 had prior ischemic heart disease requiring angioplasty orcoronary artery bypass grafts and 2 had prior myocardial infarctions butdid not receive angioplasty or coronary artery bypass grafts. In thesubgroup of 23 patients with no prior history of ischemic heart disease(using a total outcome score of ≧9 and a ≧4.7% increase in Ischemia Testvalues (i.e., absorbance associated with unbound excess metal ion)either one or three hours after exercise as positive for ischemia) therewere 2 true positives, 15 true negatives, 6 false positives and 0 falsenegatives for a sensitivity of 100% and a specificity of 72%.

Using the same criteria for positive exercise thallium treadmill andIschemia Test results, the entire 48 patients (including patients withand without a prior history of ischemic heart disease) had 6 truepositives, 29 true negatives, 11 false positives and 2 false negativesfor a sensitivity of 75% and a specificity of 73%.

Changing the positive criteria to a total thallium treadmill outcomescore of ≧10 and a ≧5.4% increase in Ischemia Test values one hour afterexercise for the entire 48 patients (including patients with and withouta prior history of ischemic heart disease) gave 3 true positives, 37true negatives, 7 false positives and 1 false negative for a sensitivityof 75% and a specificity of 88%.

EXAMPLE 8 Assessing Efficacy of an Angioplasty Procedure

Percutaneous transluminal coronary angioplasty (“PTCA”), also referredto as coronary artery balloon dilation or balloon angioplasty, is anestablished and effective therapy for some patients with coronary arterydisease. PTCA is an invasive procedure in which a coronary artery istotally occluded for several minutes by inflation of a balloon. Theinflated balloon creates transient but significant ischemia in thecoronary artery distal to the balloon. The result, however, is awidening of a narrowed artery.

PTCA is regarded as a less traumatic and less expensive alternative tobypass surgery for some patients with coronary artery disease. However,in 25 to 30 percent of patients, the dilated segment of the arteryrenarrows within six months after the procedure. In these cases, eitherrepeat PTCA or coronary artery bypass surgery is required. Additionally,complications from angioplasty occur in a small percentage of patients.Approximately, 1 to 3 percent of PTCA patients require emergencycoronary bypass surgery following a complicated angioplasty procedure.

The present invention addresses both problems by providing a means formonitoring on-going angioplasty procedures and by providing a mechanismfor monitoring the post-angioplasty status of patients.

Twenty-eight patients already scheduled for emergent or electiveangioplasty had blood samples (20 ml) drawn just prior to undergoingPTCA (“baseline”) at 6, 12 and 24 hours after the last balloondeflation, and three tubes (25 ml) at 1 minute and 6 minutes after thelast balloon deflation. Collection and administration of the test was asdescribed in Example 5 herein. A detailed description of the angioplastyprocedure was also recorded so the magnitude of ‘downstream’ ischemiacould be estimated. This included catheter size, number of inflations,inflation pressure, duration of inflation, number of vessels involvedand location.

The eligible patient population consisted of male or female patients whomet all of the following criteria: (1) 18 years or older; (2) referredfor PTCA for reasons independent of the study; (3) able to give written,informed consent; and (4) and did not possess any of the exclusionarycriteria. Patients were excluded if they met any of the followingcriteria: (1) patients who were to have PTCA performed with a perfusioncatheter; (2) patients with known, concurrent ischemic diseaseincluding, but not limited to transient ischemic attacks, cerebralvascular accident, acute myocardial infarction and intermittentclaudication. Prior to PTCA, a pretreatment evaluation was conductedwhich included documentation of all concurrent medications and thetaking of a blood sample for ischemia test administration and baseline(this occurred after the patient had been heparinized and the sheathplaced).

The standard PTCA protocol was followed at all times. In no instance wasthe drawing of the additional tubes of blood permitted to subject thepatient to additional risk (beyond the drawing of the blood), or modifythe standard protocol.

The “standard” PTCA protocol generally comprised the following: Thepatient was transported to the cardiac catheterization laboratory in thefasting state. The right groin draped and prepped in the usual sterilefashion. Local anesthesia was administered consisting of 2% lidocaineinjected subcutaneously and the right femoral artery entered using an 18gauge needle, and an 8 French arterial sheath inserted over a guide wireusing the modified Seldinger technique. Heparin, 3000 units, wasadministered I.V. Left coronary cineangiography was performed usingJudkins left 4 and right 4 catheters, and left ventricularcineangiography performed using the automated injection of 30 cc ofradiocontrast material in the RAO projection. After review of thecoronary angiography, PTCA was performed.

The diagnostic cardiac catheter was then removed from the femoral sheathand exchanged for a PTCA guiding catheter which was then positioned inthe right or left coronary ostia. An additional bolus of intravenousheparin, 10,000 units, was administered. A coronary guidewire, usually a0.014 inch flexible tipped wire, was then advanced across theobstruction and positioned distally in the coronary artery. Over thisguidewire, the balloon inflation system was inserted, usually consistingof a “monorail” type balloon dilation catheter. Sequential ballooninflations were made, with angiographic monitoring between inflations.The duration of the inflations varied among operators, but averagedapproximately 45-60 seconds; occasionally prolonged inflations between 3and 15 minutes were performed.

When it was determined that adequate opening of the coronary stenosishad been achieved, the balloon catheter was fully withdrawn and coronaryangiograms performed with and without the guidewire in position. If nofurther intervention was believed to be necessary, the sheath was thensewn into position and the patient transported to either the intensivecare unit or observation unit. The sheath was removed afterapproximately 6 hours and firm pressure applied with a C clamp or manualpressure. The patient remained at bed rest for approximately 6 hoursafter sheath removal.

Standard patient follow up was conducted according to clinical practice.

As stated, sample collection and administration of the ischemia testoccurred essentially as described in Example 5 herein. The testtechnician was masked to the time the PTCA sample was taken.

Compared to baseline, 26 of the 28 tested patients demonstratedincreased ischemia values after balloon inflation. The remaining twopatients registered false negatives, both of which started with baselinevalues above 0.400. The mean increase in the ischemia test value frombaseline to balloon inflation was 15.2%. Of the 21 patients that had 5hour samples tested, all but three demonstrated a decreased ischemiatest value compared to that measured during balloon inflation. Studyresults demonstrated that the ischemia test marker rises almostimmediately following controlled onset of ischemia during theangioplasty procedure. The rapid rise of the marker during ballooninflation and its descent over a five hour period correlated with thecontrolled start and stop of ischemia. The diagnostic accuracy of thestudy was 96 percent.

EXAMPLE 9 Evaluation of Post-Myocardial Infraction Patients

In a second study, three subsets of patients—patients without acutemyocardial infarction (NonAMI), patients with acute myocardialinfarction (AMI), and patients without AMI with significant collateralcirculation (NonAMI collateral)—all of whom were undergoing emergent orelective angioplasty had blood samples collected prior to PTCA,immediately after balloon deflation, 6 hours after the procedure, and 24hours after the procedure. A total of 63 patients were tested. Thestandard PTCA protocol (as described in Example 8) was followed.

During PTCA, blood was drawn into a syringe and then transferred tosodium-heparinized tubes. Post PTCA samples were drawn into green topsodium-heparinized tubes. In all other regards, sample collection andadministration of the ischemia test occurred essentially as described inExample 5 herein. The test technician was masked to the time the PTCAsample was taken.

The ischemia test was considered positive if it increased betweenbaseline and immediately after balloon angioplasty. The results of thestudy showed a statistically significant rise (p=0.0001) in the ischemiatest marker following balloon angioplasty and a return to baselinewithin 24 hours. The mean percent increase for all patients in the studywas 9.4%.

MEAN MEAN % TIME DIFF FROM DIFF FROM P- POINT N MEAN SD BASELINE SDBASELINE SD VALUE Baseline 62 .354 .0424 — — — — — Immed. 63 .385 .0411.0310 .0382 9.4% .1178 .0001 post PTCA 6 hours 57 .368 .0513 .0150 .05055.0% .1507 .0167 post PTCA 24 hours 43 .363 .0474 .0090 .0444 3.2% .1312.1221 post PTCA % CHANGE FROM WITH AMI WITHOUT AMI T-TEST BASELINE NMEAN SD N MEAN SD P Immed Post 19 .083 .137 41 .101 .111 .0001 PTCA 6hrs Post PTCA 15 .091 .137 39 .027 .153 .2676 24 hrs Post PTCA 14 .130.158 27 .019 .081 .2240

A side branch occlusion (“SBO”) occurs when, as a result of ballooninflation, a side artery becomes obstructed, causing loss of blood flowand ischemia distal to the occlusion. Patients with side branchocclusion (SBO) were predicted to have more ischemia than those without.Patients were assigned to the SBO subset if their cardiologist indicatedthey had significant SBO.

Study results showed significantly higher ischemia test valuesimmediately after and 6 hours after PTCA in patients with SBO. Thefollowing data includes patients in all study subsets. The number ofpatients varies because investigators were not always able to obtainblood samples at all four draw times.

% CHANGE FROM WITH SBO WITHOUT SBO T-TEST BASELINE N MEAN SD N MEAN SD PImmed Post PTCA 8 .228 .144 51 .076 .102 .0005 6 hrs Post PTCA 8 .150.156 45 .033 .149 .0480 24 hrs Post PTCA 8 .168 .222 33 .013 .098 .1500

EXAMPLE 10 Assessment of the Patency of In-situ Coronary Stent

Coronary stents may be inserted during angioplasty and left in place ona permanent basis in order to hold open the artery and thus improveblood flow to the heart muscle and relieve angina symptoms. Stentinsertion consists of the insertion of a wire mesh tube (a stent) toprop open an artery that has recently been cleared using angioplasty.The stent is collapsed to a small diameter, placed over an angioplastyballoon catheter and moved into the area of the blockage. When theballoon is inflated, the stent expands, locks in place and forms a rigidsupport to hold the artery open.

Stent use has increased significantly in just the past year, and is nowused in the vast majority of patients, sometimes as an alternative tocoronary artery bypass surgery. A stent may be used as an alternative orin combination with angioplasty. Certain features of the artery blockagemake it suitable for using a stent, such as the size of the artery andlocation of the blockage. It is usually reserved for lesions that do notrespond to angioplasty alone due to the reclosure of the expandedartery.

In certain selected patients, stents have been shown to reduce therenarrowing that occurs in 30B40 percent of patients following balloonangioplasty or other procedures using catheters. Stents are also usefulto restore normal blood flow and keep an artery open if it has been tornor injured by the balloon catheter.

However, reclosure (referred to as restenosis) is a common problem withthe stent procedure. In recent years doctors have used stents coveredwith drugs that interfere with changes in the blood vessel thatencourage reclosure. These new stents have shown some promise forimproving the long-term success of this procedure. Additionally, after astent procedure has been done, patients are often placed on one or moreblood thinning agents such as aspirin, Ticlopidine and/or Coumadin inorder to prevent or prolong reclosure. Whereas aspirin may be usedindefinitely; the other two drugs are used only for four to six weeks.

The present invention provides a mechanism for monitoring thefunctioning and patency of an in situ stent.

Stent patency was tested in the same study and same patient group inwhich post-myocardial infarction patients were studied (see Example 9).The study results showed significantly lower ischemia test valuesimmediately after and 6 hours after PTCA for those patients with stents.The following data includes patients in the NonAMI subset only. Thenumber of patients varies because investigators were not always able toobtain blood samples at all four draw times.

% CHANGE FROM WITH STENT WITHOUT STENT T-TEST BASELINE N MEAN SD N MEANSD P Immed Post 37 .089 .105 4 .210 .117 .0373 PTCA 6 hrs Post PTCA 36.009 .139 3 .243 .153 .0087 24 hrs Post PTCA 26 .022 .080 1 .071 NA NA

EXAMPLE 11

Diagnosis and Assessment of Arrhythmic/Dysrhythmic Patients

The present invention provides a rapid method for assessing arrhythmiasand diagnosing and measuring dysrhythmias.

Rapid assessment and treatment of arrhythmias is key to a successfuloutcome: if treated in time, ventricular tachycardia and ventricularfibrillation can be converted into normal rhythm by administration of anelectrical shock; alternatively, rapid heart beating can be controlledwith medications which identify and destroy the focus of the rhythmdisturbances. If an arrhythmia is not promptly diagnosed and treated, astroke may be the likely result. Arrhythmia prevents the heart fromfully pumping blood out of the heart chambers; the undisgorged bloodremaining in the heart chamber will pool and clot. If a piece of theblood clot in the atria becomes lodged in an artery in the brain, astroke results. About 15 percent of strokes occur in people with atrialfibrillation.

Traditionally, electrocardiography, also called ECG or EKG, is used todiagnosis the occurrence of an arrhythmia. (Also utilized are the “12lead EKG” and signal-averaged electrocardiogram (S.A.E.C.G.), theS.A.E.C.G. to identify people who have the potential to experience adangerous ventricular arrhythmia and the “12 lead EKG” primarily inpeople undergoing arrhythmias.) However, all of the electrocardiographictests yield frequent false positive and false negative results. Thepresent invention provides a method for supplementing all of theaforementioned electrocardiographic tests in order to reduce, if notavoid entirely, the frequency of false positive and false negativediagnoses.

Other diagnostics techniques typically used are invasive and thuspossess greater risk. For instance, transesophageal echocardiography(T.E.E.) is an imaging procedure, in which a tube with a transducer onthe end of it is passed down a person's throat and into the esophagus;images from TEE can give very clear pictures of the heart and itsstructures. Cardiac catheterization is another invasive procedure whichallows for measurement and viewing of the pumping ability of the heartmuscle, the heart valves and the coronary arteries. The shortcoming ofthese procedures, however, lies in their invasive nature.

The present invention provides a non-invasive method for diagnosis andmeasurement of dysrhythmias which can be used in lieu of, or insupplementation of, the aforementioned invasive procedures.

Patients with dysrhythmias undergoing PTCA were predicted to have moreischemia than those without. (Dysrhythmia is cited in the medicalliterature as a good indicator of ischemia.) In the 63 patient studydetailed in Examples 9 and 10, patients were additionally assigned to adysrhythmia subset if their medical record showed significantdysrhythmia during PTCA. Study results showed significantly higherischemia test values immediately after and 6 hours after PTCA inpatients with significant dysrhythmias. The following data includespatients in all study subsets. The number of patients varies becauseinvestigators were not always able to obtain blood samples at all fourdraw times.

WITH W/O % CHANGE FROM DYSRHYTHMIA DYSRHYTHMIA T-TEST BASELINE N MEAN SDN MEAN SD P Immed Post 5 .265 .151 57 .079 .103 .0004 PTCA 6 hrs PostPTCA 5 .204 .175 51 .035 .141 .0150 24 hrs Post PTCA 5 .144 .236 37 .017.107 .3000

EXAMPLES 12-23 Use of N-terminus Peptide Probe in the Evaluation ofIschemia

Under the present invention, an amino acid sequence found within theN-terminus sequence of albumin is required for cobalt binding. Thissequence has been identified as Asp-Ala-His-Lys (abbreviated “DAHK”,residues 1-4 of SEQ. ID. NO. 1). The binding characteristics of thistetrapeptide have been extensively studied and it has been determinedthat this tetrapeptide may be used to detect the presence of ischemia.

Specifically, a biological sample containing albumin is contacted withCoCl₂.6H₂O. Some of this cobalt will bind to albumin. The remaining freecobalt is then reacted with a known amount of D-A-H-K-R (residues 1-4 ofSEQ. ID. NO. 1) added to the biological sample, wherein R is anychemical group or enzyme, including no group at all or a fluorescentgroup, capable of being detected. Because D-A-H-K-R (residues 1-4 ofSEQ. ID. NO. 1) has a great affinity to cobalt (association constantabout 10¹⁵) the free cobalt will attach to it. The D-A-H-K-R (residues1-4 of SEQ. ID. NO. 1) differs from Co-D-A-H-K-R (residues 1-4 of SEQ.ID. NO. 1) spectroscopically. One distinction is that Co-D-A-H-K-R(residues 1-4 of SEQ. ID. NO. 1) has an extinction coefficient that is1.5 to 2 times the peptide alone. This phenomenon can be used todetermine that the peptide has bound to the cobalt (an increase inabsorption at about 214 nm using HPLC or other methods).

EXAMPLE 12

To a 0.2 ml sample of blood or plasma was added 50 μL 0.1% CoCl₂. Themixture was incubated for 5 to 10 minutes. Thereafter, 50 μL of 1 mg/mlof D-A-H-K-R was added to the sample. (R was a polymer or othersubstance having chemical and physical characteristics that changed whenthe cobalt binds to the peptide—causing a small current change or anyother change that was detected.) The sample was then centrifuged(Centricon 10 or 3) for 5 minutes, followed by HPLC analysis of thefiltrate using a ultrahydrogel 120, 5μ column at 60° C.; isocratic run,mobile phase acetonitrile: ammonium acetate buffer 30 mM pH 8.0, 2:98;at 1 ml/minute and U.V. detection at 214 nm. The peptide peak appearedat about 5.88 minutes.

The same procedure was run with a peptide control (no cobalt). Thedifference in peak size between test (with cobalt) and control (nocobalt) was proportional to the amount of free cobalt and henceischemia.

The following preliminary experiments illustrate the properties andcritical characteristics of the peptide probe.

EXAMPLE 13

Measurement of Cobalt Binding to HSA and Octapeptide using Cold CobaltBinding Assay

OBJECTIVE: To investigate cobalt binding to the octapeptide and humanserum albumin using cold cobalt binding assay.

EXPERIMENTAL: Octapeptide synthesized at the Inorganic ChemistryDepartment (BAM 1, Pat Ingrey, Cambridge):NH₂-Asp-Ala-His⁺-Lys⁺-Ser-Glu-Val-Ala-CONH₂, residues 1-8 of SEQ. ID.NO. 1) Molecular weight: 855.4 Da.

SOLUTIONS: CoCl₂ 0.1% (w/v)=4.2 mM; HSA 3% (w/v)(in 75 mM HEPES pH7.4)=0.45 mM; Octapeptide 0.965 mM (in 75 mM HEPES pH 7.4); HEPES 75 mMpH 7.4; DTT 0.15% (w/v); NaCl 0.85% (w/v).

METHOD: Fifty μL 0.1% CoCl₂ was added to tubes each containing 200 μL of75 mM HEPES pH 7.4 or 0.45 mM HSA in HEPES or 0.965 mM Peptide in HEPES;the tubes were allowed to stand at room temperature for 10 minutes; 50μL DTT 0.15% was added to one tube (test tube) and distilled H₂O to theother (control tube); the tubes were maintained for 2 minutes at roomtemperature; 1 ml NaCl 0.85% was then added; the absorbance at A470 nmof the test tube versus the blank was measured.

RESULTS:

mean ID A470 nm A470 % bound 75 mM HEPES pH 7.4 1.087 1.083 1.085 0.00.45 mM HSA in HEPES pH 7.4 0.668 0.643 0.656 39.5 0.965 mM Pepticle inHEPES pH 7.4 0.638 0.655 0.647 40.4

CONCLUSIONS: Under the conditions used for the binding measurements,this experiment showed that: 1. Cobalt binds to the “oetapeptide”(N-Asp-Ala-His⁺-Lys⁺-Ser-Glu-Val-Ala, residues 1-8 of SEQ. ID. NO. 1);2. However, the octapeptide (0.965 mM) binds cobalt with a stoichiometryof 1:2.3.

EXAMPLE 14 Mass Spectrometry of Octapeptide After the Addition of Cobalt

OBJECTIVE: To investigate whether mass spectral study would providemolecular weight information for the octapeptide and its correspondingcobalt complex.

SOLUTIONS: Ammonium acetate 20 mM-pH 7.4 (with dilute ammonia solution);CoCl₂20 μM (in HPLC grade H₂O); Octapeptide 9.5 μM (in HPLC grade H₂O).

METHOD: 20 μM CoCl₂ (100 μl) was added to 9.5 μM octapeptide (100 μl )and mass spectrometry carried out.

RESULTS: The main molecular ion peak was observed at 855.4 Da, withminor peaks at 877.4 and 893.4 Da probably as a result of sodium andpotassium cluster ions After the addition of cobalt, an extra molecularion peak was observed at 912.3 Da.

CONCLUSIONS: Octapeptide showed a molecular ion at 855 Da consistentwith the expected molecular weight of the peptide moiety. Octapeptideplus cobalt complex showed a molecular ion at 912 Da suggesting that atleast two protons are removed during the complex formation.

EXAMPLE 15 Spectrophotometric Analysis of the Octapeptide andOctapeptide-Cobalt Complex

OBJECTIVE: It is clear from the previous mass spectrometry evidence thatcobalt forms a complex with the octapeptide with a concomitant loss oftwo possible protons. Metal complexes in general show distinctabsorption in the UV range and in many cases these complexes show eithera hypochromic or a bathochromic shift in the spectra. These shifts canbe correlated to provide the energy of binding. It was thereforeanticipated that the octapeptide-cobalt complexation would provide suchinformation.

METHOD: The quartz cuvette contained 800 μl octapeptide+200 μl H₂O(control) or CoCl₂ (complex). Spectra were run from 180 to 800 nm on asingle beam spectrophotometer.

CONCLUSIONS: Cobalt and octapeptide individually have peak absorbancesat <200 and 225 nm respectively with little overlap. Following additionof a CoCl₂ solution to octapeptide (1.1:1) there was no significantshift in the K_(max) (220 nm). The absorption band at this regionbroadened indicating complex formation, but the result could not be usedto determine the binding energy (constant).

EXAMPLE 16 Mass Spectrometry of Octapeptide After the Addition of Cobalt

OBJECTIVE: To investigate whether mass spectral study would providemolecular weight information for the peptide and its correspondingcobalt complex.

METHOD: 20 or 200 μM CoCl₂ (100 μl) was added to 22.9 μM octapeptide(100 μl) to give ratios of cobalt:octapeptide of 1:1.1 and 8.7:1respectively. Mass spectra for the two samples were carried out as perconditions detailed in the previous experiment.

RESULTS: One major molecular ion peak was observed at 855.4 Darepresenting the octapeptide alone. After the addition of 20 μM cobaltto the octapeptide, two peaks were observed, a major peak at 855.3representing octapeptide only plus a minor peak at 912.2 Da representingoctapeptide-cobalt complex. Peak ratio of free octapeptide tooctapeptide-cobalt complex was 1:0.15. A similar profile was observedfollowing the addition of 200 μM cobalt to the octapeptide. Peak ratioof free octapeptide to octapeptide-cobalt complex was 1:0.9.

CONCLUSIONS: On addition of cobalt (59 Da) to the octapeptide, themolecular ion peak should have occurred at 914 Da. The actual peakoccurred at 912 Da, representing the loss of two protons. On addition ofincreasing concentrations of cobalt the peak ratio of free octapeptideto octapeptide-cobalt complex increased.

EXAMPLE 17 The Effect of Oxygen on the Binding Capacity of Octapeptidefor Cobalt

OBJECTIVE: Previous experiments have highlighted the requirement ofoxygen in promoting cobalt binding to HSA. It may be anticipated thatsimilar effects could be observed in the manner of cobalt binding to theoctapeptide.

METHOD: Octapeptide-cobalt complex (no oxygen): HPLC grade H₂O wasbubbled with 100% helium for 10 minutes prior to use and used to preparethe above solutions. These were further deoxygenated for 10 minutesbefore adding 200 μM CoCl₂ (2 ml) to 22.9 μM octapeptide (2 ml). Thismixture was again deoxygenated for 10 minutes prior to analysis by HPLC.

Octapeptide-cobalt complex (with oxygen): HPLC grade H₂O was bubbledwith 100% oxygen for 10 minutes prior to use and used to prepare theabove solutions. These were further oxygenated for 10 minutes beforeadding 200 μM CoCl₂,(2 ml) to 22. μM octapeptide (2 ml ). This mixturewas again oxygenated for 10 minutes prior to analysis by HPLC.

HPLC Analysis: Chromatography was carried out on a KS437 styrene/DVBpolymer column (4.6 mm×150 mm, pore diameter 100-150 A, BioDynamics)under isocratic conditions of 2% acetonitrile in 30 mM Ammonium acetatepH 8.0 at a flow rate of 2 ml/min. Peaks were detected at 230 nm.Chromatography gave two distinct peaks at 230 nm, the first peakrepresenting octapeptide-cobalt complex and the second peak representingfree octapeptide. Octapeptide-Co²⁺ complex formed in the presence ofoxygen gave a higher ratio of complex over free peptide, as indicated bythe first peak being the larger of the two. Octapeptide-Co²⁺ complexformed in the absence of oxygen again gave two peaks but the second peakwas now the larger of the two, indicating less complex formation.CONCLUSIONS: It would appear that oxygenated conditions enhance cobaltbinding to the octapeptide.

EXAMPLE 18 The Effect of pH on the Octapeptide

OBJECTIVE: To optimize chromatography conditions for analysis ofoctapeptide by HPLC.

METHOD: The octapeptide was analyzed by HPLC using a KS437 styrene/DVBPolymer column (4.6 mm×150 mm, pore diameter 100-150 A, ‘BioDynamics)under isocratic conditions of 2% acetonitrile in 30 mM Ammonium acetateat pH 6.2, 7.5 and 8.0 at a flow rate of 2 ml/min. Peaks were detectedat 230 nm.

RESULTS: At pH 6.2, the octapeptide eluted after 1.6 min. At pH 8.0 theretention time had increased to 2.1 min. When the octapeptide was run atpH 7.5, two peaks were observed at 1.6 and 2.1 min.

CONCLUSIONS: The octapeptide exists in two forms depending on pH. Theprotonated form elutes at pH 6.2, and the deprotonated form at pH 8.0.

EXAMPLE 19 The Effect of pH on the Binding of Cobalt to the Octapeptide

OBJECTIVE: It was reported that the peptide peak ‘shifted’ when asolution of cobalt chloride was added to the octapeptide. It was decidedto investigate this phenomenon fully as this would provide a direct toolfor the determination of several parameters of cobalt binding to theoctapeptide.

METHOD: 200 mM CoCl₂ (30 μl) was added to 2.3 mM octapeptide (270 μl),incubated at room temperature for 10 minutes and analyzed by HPLC. HPLCanalysis: The octapeptide-cobalt complex was analyzed by HPLC using aKS437 styrene/DVB polymer column (4.6 mm×150 mm, pore diameter 100-150A, BioDynamics) under isocratic conditions of 2% acetonitrile in 30 mMAmmonium acetate at pH 6.2 and 8.0 at a flow rate of 2 ml/min. Peakswere detected at 230 nm.

RESULTS: At pH 6.2, a single peak eluted after 1.6 min in the presenceand absence of cobalt. At pH 8.0 however a single peak eluted after 1.2min in the presence of cobalt and at 2.1 min in the absence of cobalt.

CONCLUSIONS: The octapeptide exists in two forms depending on pH. Theprotonated form that elutes at pH 6.2 is unable to bind cobalt andtherefore its elution profile is unchanged. In contrast, thedeprotonated form which exists at pH 8.0 is able to bind cobalt,resulting in an increased UV absorption and a decreased retention time,1.2 min as opposed to 2.1 min for the free octapeptide.

EXAMPLE 20 The Titration of Octapeptide with Increasing Concentrationsof Cobalt

OBJECTIVE: To determine whether increasing concentrations of cobaltresulted in a sponding increase in octapeptide-cobalt complex formation.

METHOD: Octapeptide was used at a final concentration of 2.1 mMthroughout, with asing concentrations of CoCl₂, as shown in the Tablebelow:

[Octa- Vol Ratio of Vol CoCL₂ peptide] octapeptide octapeptide:[CoCL₂](mM) added (μl) (mM) added (μl) CoCL₂ 0 0 2.3 27 1:0 1 3 2.3 2721:1  1.25 3 2.3 27 16.8:1   2.25 3 2.3 27 9.3:1   4.5 3 2.3 27 4.7:1  10 3 2.3 27 2.1:1   18 3 2.3 27 1.2:1   36 3 2.3 27   1:1.7 72 3 2.3 27  1:3.4 200 3 2.3 27   1:9.5

HPLC analysis: The octapeptide-cobalt complex was analyzed by HPLC usinga KS437 styrene/DVB polymer column (4.6 mm×150 mm, pore diameter 100-150A, BioDynamics) under isocratic conditions of 2% acetonitrile in 30 mMAmmonium acetate at pH 8.0 at a flow rate of 2 ml/min. Peaks weredetected at 230 nm.

RESULTS: Mean % Peak Height:

Peak 1 Final (Octapeptide-Co Peak 2 Peak 3 [CoCL₂] (mM) complex)(unknown) (Octapeptide) 0 — 3.72 96.28 0.1 7.44 7.08 85.49 0.125 9.797.55 82.66 0.225 15.65 15.66 68.52 0.45 25.36 19.67 54.98 1.0 58.66 —50.42 1.8 61.19 14.97 23.85 3.6 69.55 13.69 16.76 7.2 71.49 14.47 14.0520.0 82.17 10.27 7.56

From the table immediately preceding, a plot of Log cobalt concentrationversus % peak height for peak 3 was produced using Prism software. The50% binding constant as deduced from the exponential graph had a valueof 0.6461 mM.

CONCLUSIONS: For 50% binding, 0.6461 mM Co²⁺ binds to 2.1 mMoctapeptide. Therefore for 100% binding, 1.2922 mM Co²⁺ binds to 2.1 mMoctapeptide. The stoichiometry of cobalt binding to octapeptide is 0.615cobalt to 1 octapeptide.

EXAMPLE 21 Liquid Chromatography-Mass Spectrometry of Octapeptide Afterthe Addition of Cobalt

OBJECTIVE: To investigate whether mass spectral study would providemolecular weight information for the peptide and its correspondingcobalt complex.

METHOD: 200 mM CoCl₂ or H₂O (3 μl) was added to 2.3 mM octapeptide (27μl) and incubated at room temperature for 10 minutes. LC-MS analysis:Liquid chromatography was performed using a KS437 styrene/DVB polymercolumn (4.6 mm×150 mm, pore diameter 100-150 A, BioDynamics) underisocratic conditions of 2% acetonitrile in 30 mM Ammonium acetate at pH8.0 at a flow rate of 0.5 ml/min. Peaks were detected at 230 nm, andanalyzed by on line mass spectrometry.

RESULTS: In the control sample, two molecular ion peaks were observed at855.2 Da, representing the octapeptide alone, and at 877.2 Da,representing an octapeptide-sodium cluster. After the addition of 200 mMcobalt, one major peak was observed at 911.1 Da.

CONCLUSIONS: On addition of cobalt (59 Da) to the octapeptide, themolecular ion peak should occur at 914 Da. The actual peak occurs at 911Da, representing the loss of protons.

EXAMPLE 22 Endprotease Lys-C Digest of Octapeptide and its SubsequentIncubation with Cobalt

OBJECTIVE: Previous experiments confirm that CoCl₂ forms a stablecomplex with the octapeptide. In order to elucidate the site ofattachment, the octapeptide was cleaved stereoselectively with theendoprotease Lys-C. The resultant tetrapeptides upon incubation withCoCl₂ would allow elucidation of the probable binding site.

METHOD: Octapeptide 1.97 mg/ml (250 μl) was incubated with theendoprotease Lys-C 100 μg/ml (50 μl ) at a substrate:enzyme ratio of100:1 (w/w) in 8.3 mM Tricine, 1.6 mM EDTA pH 8.0 at 37° C. for 24 h.After digestion, 27 μl of the product was incubate with 200 mM CoCl₂ (3μ) at 20° C. for 10 minutes prior to analysis by HPLC. HPLC Analysis:The products from the Lys-C digest were analyzed by HPLC using an aminocolumn (4.6 mm×250 mm. pore diameter 100 Å, BioDynamics-73) underisocratic conditions of 30 mM Ammonium acetable at pH 8.0 at a flow rateof 1.5 ml/min. Peaks were detected at 230 nm.

RESULTS: When the digested Lys-C products were run on HPLC, two peakswere observed at 2.6 and 8.9 min, designated tetrapeptides 1 and 2respectively. Similarly after addition of cobalt to the digestedproducts two peaks were again observed. However, tetrapeptide 1exhibited an increased UV absorption and decreased retention time,eluting at 1.7 min as opposed to 2.6 min.

CONCLUSIONS: The octapeptide was digested at the C terminus of thelysine residue by the endoprotease yielding two tetrapeptides. Onaddition of cobalt to the endoprotease digested octapeptide, a singletetrapeptide-cobalt complex was formed with tetrapeptide 1. Thereappeared to be no effect on tetrapeptide 2.

EXAMPLE 23 Mass Spectrometry Analysis of the Tetrapeptide 1-CobaltComplex

OBJECTIVE: To determine the identity of tetrapeptide 1.

EXPERIMENTAL: Tetrapeptides 1 and 2 were fractionated by HPLC andcollected. CoCl₂ 1.2 mM (3 μl) was added to tetrapeptide 1 (27 μl) andincubated at room temperature for 10 minutes. Samples were subsequentlyrun on MS as described previously.

RESULTS: Tetrapeptide 1 gave two molecular ion peaks at 470.1 and 477.1Da. Tetrapeptide 2 gave a single peak at 404.0 Da. Tetrapeptide 1-cobaltcomplex gave two peaks at 477.1 and 526 Da.

CONCLUSIONS: Tetrapeptide 1 is determined to be Asp-Ala-His-Lys(residues 1-4 of SEQ. ID. NO. 1) with a molecular weight of 469 Da.Tetrapeptide 2 is determined to be Ser-Glu-Val-Ala (404 Da, residues 5-8of SEQ. ID. NO. 1). Cobalt binds to Asp-Ala-His-Lys (residues 1-4 ofSEQ. ID. NO. 1) forming a complex of 526 Da with a loss of 3 protons.The molecular ion peak observed at 477.1 Da is a contaminant from theLys-C preparation.

EXAMPLE 24 Manufacture of Calibrator Solutions

Human albumin solutions of 35 mg/ml containing cobalt of molar ratios of0, 0.4, 0.625, 0.83, 1.25 and 2.5 to 1, cobalt:albumin, were madeaccording to the following protocol.

An albumin solution of 35 mg/ml, Solution A, was made by initiallydissolving 40 g solid human albumin (Fraction V, Sigma Chemical Co., St.Louis) in 900 ml 50 mM Tris-Cl, pH7.2, 0.15 NaCl, and assessing albuminconcentration with bromo cresol green (BCG) assay (Sigma Chemical Co.).Additional buffer was added to produce an albumin concentration of 35mg/ml. This solution was allowed to sit at 4° C. for at least 24 hoursprior to use.

To 500 ml of Solution A, 1.27 ml 0.32M Co(OAc)₂.6H₂O (160 mg Co salt/2ml H₂O) (Sigma Chemical Co.) was added drop-wise with gentle swirling toproduce a cobalt:albumin molar ratio of 1.25:1, Solution B. Thissolution was allowed to sit at room temperature for one hour prior tostorage at 4° C. until use.

Different volumes of Solutions A and B were mixed to produce additionalcalibrator solutions:

Cobalt:Albumin ratio Solution A, ml Solution B, ml 0 200 0 0.4 133 670.625 100 100 0.83 67 133 1.25 0 200

To make a cobalt:albumin calibrator solution of 2.5:1, 0.94 ml of 0.32MCo(OAc)₂ was added to 229 ml of Solution A. This solution was permittedto sit at room temperature for one hour and then stored at 4° C. untiluse.

EXAMPLE 25 Quality Control Characterization of Calibrator Solutions

To obtain a cobalt:albumin ratio, one ml aliquots of each of the fivecalibrator solutions (each of which had been in storage for 24 hoursprior to testing) was placed individually in dialysis bags and dialyzedagainst 400 ml 50 mM Tris-Cl, pH7.2, 0.15M NaCl, with three changes ofbuffer at room temperature. Three to 5 μl of the dialyzates werewithdrawn and analyzed for albumin using 1 ml of the BCG dye from SigmaChemical Co. Absorbance was read at 628 nm after 30 seconds.

Cobalt was assessed by atomic absorption by Galbraith Laboratories,Inc., Knoxville, Tenn.

The cobalt:albumin ratios were found to conform to expected values forall five calibrator solutions.

Added Cobalt, Co:albumin At equilibrium, Co:albumin 0.4 0.16 0.625 0.260.83 0.31 1.25 0.46 2.50 0.74

These results indicate that the amount of cobalt bound per albuminmolecule following dialysis remained proportional to the original metalconcentration in the calibrator solution, indicating that themetal-cobalt complex is stable.

EXAMPLE 26

Generating a Standard Curve Using Calibrator Solutions

Aliquots of 200 μl were withdrawn from each calibrator solution storedat 4EC into 12×75 mm borosilicate tubes and allowed to equilibrate toroom temperature for at least 15 minutes.

A standard solution of 0.8% CoCl₂.6H₂O in H₂O had been made bydissolving 0.4 g solid in 500 ml deionized H₂O in a 500 ml polystyrenebottle; cobalt concentration was confirmed by atomic absorption byGalbraith Laboratories, Inc. Fifty μl of 0.8% CoCl₂ solution was addedto each calibrator solution and gently mixed.

A 10 mM DTT standard solution had been made by equilibrating the bottleof DTT (DL-dithiothreitol, Sigma Chemical Co.) to room temperature,weighing 12 mg and dissolving same in 8 ml deionized water. Thesulfhydryl content of this solution was assessed using Ellman's Reagent,5,5′-thio-bis(2-nitrobenzoic acid), Sigma Chemical Co. Exactly 10minutes after addition of CoCl₂ solution to the calibrator solutions, 50μl of the 10 mM DTT solution was added, mixed and allowed to react for 2minutes. Substitution of DTT with 50 μl 0.9% NaCl was used as the blank.The reaction was quenched by the addition of 1.0 ml 0.9% NaCl.Absorbance at 470 nm on day 1 was read as soon as practicable.Absorbance was read again on days 12, 20 and 23:

Calibrator A470 A470 A470 A470 Co:albumin Day 1 Day 12 Day 20 Day 23 00.26 0.26 0.23 0.27 0.4 0.32 0.30 0.28 0.29 0.625 0.33 0.33 0.31 0.311.25 0.39 0.40 0.37 0.37 2.5 0.64 0.60 0.60 0.57

Absorbance was plotted against metal concentration originally present inthe calibrator solution. The plot was found to be substantially linearover the period studied.

EXAMPLE 27 The NMR Spectra for the Complex of Ni and Albumin N-terminalAmino Acids

Addition of cobalt or nickel chloride to the synthetic albuminN-terminus octapeptide afforded changes in the appearance of the ¹H-NMRspectrum for the resonances of the first three amino acid residues, withdiagnostic changes of the Ala-2 methyl doublet at 1.35 ppm. Titrationwith NiCl₂ gave a sharp diamagnetic ¹H-NMR spectrum, while addition ofCoCl₂ induced paramagnetism at the binding site resulting in significantbroadening to the resonances associated with the three residues boundaround the metal sphere. FIG. 4 shows selected regions of the ¹H-NMRspectra (500 MHz, 10% D₂O in H₂O, 300K) showing the Ala resonances(Ala-2 and Ala-8) of the octapeptide (A) free of any metal, with a Lys-4methylene resonance appearing between the doublets for Ala2 at about1.35 ppm and for Ala8 at about 1.4, (B) with 0.5 equiv. of NiCl₂ addedresulting in a shift of the Ni-bound Ala2 doublet to about 1.3, (C) with1.0 equiv. of NiCl₂ added, (D) with 0.5 equiv. of CoCl₂ added, and (e)with 1.0 equiv. of CoCl₂ added. In all cases, the appearance andchemical shift of the resonances attributed to Ser-5, Glu-6, Val-7 andAla-8 did not change significantly after metal addition (up to oneequivalent). All these observations were conserved in metal titrationexperiments with the synthetic tetrapeptide (N-Asp-Ala-His-Lys).

EXAMPLE 28 U.V. Spectroscopic Evidence of Co Binding to Albumin Pep-12Peptides

The albumin N-terminal peptideAsp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys (Pep 12, residues 1-12of SEQ. ID. NO. 1), was synthesized by Quality Controlled Biochemicals,Inc. both in N-acetylated-Asp and free Asp forms, each with freeC-terminus. Solutions of 1 mg/ml of the two peptides were made in Tris50 mM 0.9% NaCl pH7.2 and analyzed by UV spectroscopy (Ocean Optics SD2000 and AIS Model DT 1000 as light source). U.V. spectra of Pep-12 andacetylated Pep-12 are set forth in FIGS. 5A and 5B, respectively.Addition of CoCl₂.6H₂O 0.8% (20 μL of the peptide solution) shows adramatic shift of the λ maximum of the peptide peak as well as a majorincrease in the extinction coefficient for the nonacetylated Pep-12(FIG. 6A) and no change in the spectrum of the acetylated Pep-12 (FIG.6B).

Solutions of Pep-12 and acetylated Pep-12 were made into solutions of 1mg/ml in Tris 50 mM NaCl 0.9% pH7.2. Five mixtures of the two startingpeptides were made: 100% Pep-12, 75:25 Pep-12:AcPep-12, 50:50Pep-12:AcPep-12, 25:75 Pep-12:AcPep-12 and 100% AcPep-12.

1 2 3 4 5 Pep-12 20 ml 15 10 5 0 1 mg/ml AcPep-12 — 5 10 15 20 1 mg/inl+/−CoCl2 20 20 20 20 20 0.08%

Spectral analysis of solutions 1-5 is represented in FIG. 7, from whichit can be seen that Pep-12 binds cobalt, AcPep-12 does not bind cobalt.Further, as acetylation increases, cobalt binding goes down.

EXAMPLE 29 U.V. Spectroscopic Evidence of Co Binding to Albumin Pep-10

Pep-10 was made into 1 mg/ml solutions and incubated with CoCl₂ (0.08%).Spectral scans were obtained (data not shown). There was no apparentdifference in the absorbance after addition of cobalt, indicating thatPep-10 does not bind cobalt.

EXAMPLE 30 Copper/Cobalt Competition Binding for Albumin Pep-12

Pep-12 (20 μL of 1 mg/ml or 0.014 μMol) was mixed with 5 μL CuCl₂ (0.08%or 0.023 μMol) and 20 μL CoCl₂ 0.08% (0.067 μMol). The U.V. spectralcurve is shown in FIG. 8A. AcPep-12 (20 μL of 1 mg/ml or 0.014 μMol) wasalso mixed with 5 μL CuCl₂ (0.08% or 0.023 μMol) and 20 μL CoCl₂ 0.08%(0.067 μMol). The U.V. spectral curve is shown in FIG. 8B. The CuCl₂ wasadded to Pep-12 and AcPep-12 before addition of CoCl₂. No shift orchange occurred by this manipulation.

Pep-12 binds copper and cannot therefore display a shift and increaseabsorbance when cobalt is added. The tails appearing on the peaks inFIGS. 8A and 8B are due to absorbance of copper in the U.V. range.

EXAMPLE 31 Enzymatic Acetylation of N-Terminal Pep-8 and Human SerumAlbumin

Human serum albumin (Sigma A-1653) was incubated at 37° C. for 1 h withN-acetyl transferase and acetyl CoA, and spectral scans were obtained atvarious times (2-60 minutes). A steady increase at A235 was observed(assuming A235 reflects acetylation), reaching a plateau at about 40minutes (data not shown).

Likewise, Pep-8 (Asp-Ala-His-Lys-Ser-Glu-Val-Ala, residues 1-8 of SEQ.ID. NO. 1), was acetylated according to the following conditions:

1 2 3 4 5 6 7 8 Pep-8 250 μL 250 μL 250 μL 250 μL NAT 50 μL 50 μL 50 μL50 μL AcCoA 25 μL 25 μL 25 μL 25 μL Buffer 50 μL 75 μL 25 μL 300 μL 275μL 325 μL 250 μL CoCl₂ +/−50 μL +/−50 μL +/−50 μL +/−50 μL +/−50 μL+/−50 μL +/−50 μL +/−50 μL

The Pep-8 was 1 mg/ml in a solution of Tris 50 mM, pH 7.5, 0.15 NaCl.The N-acetyl-transferase was 10 U/mL (Sigma A426). The acetyl CoA was 10mg/ml in H₂O (Sigma A2056). The Buffer was Tris 50 mM, pH 7.5, 0.15NaCl. After completion of the reaction, test tubes were centrifugedusing Centricon (3000 MW cutoff) to remove N-acetyl transferase andacetyl CoA which introduce interference in the U.V. range. The +/− inthe final row refers to the fact that the absorbance at 235 was measuredwith and without addition of CoCl₂. Addition of cobalt did not result ina shift of the peak, indicating that the acetylated Pep-8 did not bindcobalt.

FIG. 9 is the subtracted scan of the centrifuged acetylated Pep-8, plusreaction mixture and cobalt, minus the reaction mixture without thecobalt, showing a peak at about 280 nm, presumably the acetylated Pep-8.

EXAMPLE 32 Confirmation of Ni, Co and Co Binding to Modified Peptides by¹H-NMR (800 MHz)

Peptide 1: The N-terminal dodecapeptide,Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys (residues 1-12 of SEQ.ID. NO. 1).

The N-terminal dodecapeptide was titrated with each of cobalt, copperand nickel. The methyl signals of the two Ala residues (positions 2 and8) appear at the same resonance, namely 1.3 ppm. FIG. 10A is Peptide 1at pH 2.55 with no metal. FIG. 10B is Peptide 1 at pH 7.33 with nometal. Titration with 0.3 equivalent NiCl₂ at pH 7.30 is characterizedby the appearance of a set of peaks at 1.25 ppm which is characteristicof the methyl of Ala at position 2 (FIG. 10C). After the addition of oneequivalent of NiCl₂ at pH 7.33, the methyl groups of Ala at positions 2(1.3 ppm) and 8 (1.25 ppm) are equivalent, showing that the metal bindsand that the binding is stoichiometric (FIG. 10D). FIG. 10 scans wereconducted at 800 MHz, 10% D₂0/90% H₂O (Ala-Me region).

The addition of CoCl₂ also shows binding but the peaks are broader witha shift in the methyl group Ala 2 to 1.7 ppm (FIG. 11). FIG. 11A showsPeptide 1's Ala2 and Ala8 methyl signals at 1.3 (pH 2.56). FIG. 11Bshows Peptide 1 at pH 7.45. FIG. 11C shows widening of the 1.3 ppm peakas 0.5 equivalent CoCl₂ is added at pH 7.11. FIG. 11D shows a separatepeak for Ala2-Me at 1.7 ppm with 1.0 equivalent CoCl₂ at pH 7.68. FIG.11 scans were conducted at 500 MHz, 10% D₂0/90% H₂O (Ala-Me region).

The addition of CuSO₄ causes even more broadening of both methyl groupsat positions 2 and 8 to the point where, after addition of 1 equivalentof CuSO₄, both signals are lost (FIG. 12). FIG. 12A shows Peptide 1 atpH 2.56 with Ala2 and Ala8 methyl signals at 1.35 ppm. FIG. 12B showsPeptide 1 at pH 7.54. FIG. 12C shows Peptide 1 with a broadening of thesignal at 1.35 ppm, due to about 0.5 equivalent CuSO₄ (pH 7.24). FIG.12D shows Peptide 1 with about 1 equivalent CuSO₄ at pH 7.27. FIG. 12scans were conducted at 500 MHz, 10% D₂0/90% H₂O (Ala-Me region).

Peptide 2: The N-Terminal dodecapeptide,Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys (residues 1-12 of SEQ.ID. NO. 1), in which the amino group of the N-terminal Asp has beenacetylated.

Addition of NiCl₂ to the acetylated derivative does not result inbinding, i.e., there is no appearance of additional peaks (FIG. 13).However, addition of even one equivalent of NiCl₂ broadens the spectrumconsiderably due to the fact that the nickel is free in solution. FIG.13A shows Peptide 2 at pH 2.63 with the Ala2 and Ala8 Me signals atabout 1.28 ppm. FIG. 13B shows Peptide 2 at pH 7.36. FIG. 13C showsPeptide 2 with about 0.5 equivalent NiCl₂ at pH 7.09. FIG. 13D showsPeptide 2 with about 1 equivalent NiCl₂ at pH 7.20. FIG. 13 scans wereconducted at 800 MHz, 10% D₂0/90% H₂O (Ala-Me region).

The N-Terminal Unodecapeptide,Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys (residues 1-11 of SEQ. ID.NO. 1), in which the terminal Asp is missing.

The N-terminal residue is Ala and consequently the position of thedoublet from the methyl group is pH dependent (FIG. 14). Addition ofNiCl₂ does not result in complex formation. FIG. 14A shows Peptide 3 atpH 2.83 with the Ala2 signal at 1.5 and the Ala8 signal at 1.3. FIG. 14Bshows Peptide 3 at pH 7.15. FIG. 14C shows Peptide 3 with 0.13equivalent NiCl₂ at pH 7.28. FIG. 14D shows Peptide 3 with about 0.25equivalent NiCl₂ at pH 7.80. FIG. 14E shows Peptide 3 with 0.5equivalent NiCl₂ at pH 8.30. FIG. 14 scans were conducted at 500 MHz,10% D₂0/90% H₂O (Ala-Me region).

Peptide 4: The N-Terminal decapeptide,His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys (residues 1-10 of SEQ. ID. NO.1), in which Asp-Ala has been removed.

Upon addition of NiCl₂ the spectrum broadens unrecognizably with noevidence of binding (FIG. 15). FIG. 15A shows Peptide 4 with an Ala8signal at 1.8 ppm at pH 2.72. FIG. 15B shows Peptide 4 at pH 7.30. FIG.15C shows Peptide 4 with 0.5 equivalent NiCl₂, pH 8.30. FIG. 15D showsPeptide 4 with about 1 equivalent NiCl₂ at pH 8.10. FIG. 15 scans wereconducted at 800 MHz, 10% D₂0/90% H₂O (Ala-Me region).

Peptide 5: The nonapeptide, Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys(residues 1-9 of SEQ. ID. NO. 1), in which the tripeptide Asp-Ala-His ismissing.

Again there is not much change in the spectrum after addition of 0.3equivalents of NiCl₂ (FIG. 16C) except for the decrease in peakintensity and peak broadening upon addition of less than 1 equivalent ofmetal ions (FIG. 16D). There is no evidence of metal binding. FIG. 16Ais Peptide 5 at pH 2.90 with the Ala8 signal at 1.3 ppm. FIG. 16B isPeptide 5 at pH 7.19. FIG. 16C is Peptide 5 with 0.3 equivalent NiCl₂,pH 7.02. FIG. 16D is Peptide 5 with about 0.6 equivalent NiCl₂ at pH7.02. FIG. 16 scans were conducted at 500 MHz, 10% D₂O/90% H₂O (Ala-Meregion).

Peptide 6: The N-terminal tetrapeptide, Asp-Ala-His-Lys, residues 1-4 ofSEQ. ID. NO. 1).

The addition of NiCl₂ (FIG. 17), CoCl₂ (FIG. 18) and CuSO₄ (FIG. 19) allgave diagnostic changes consistent with metal ion binding. The spectraresemble those obtained with the dodecapeptide (Peptide 1) and not thoseobtained with Peptides 2, 3, 4 and 5.

FIG. 17A is the N-terminal tetrapeptide at pH 2.49 with an Ala2 signalat 1.3 ppm. FIG. 17B is the tetrapeptide at pH 7.44. FIG. 17C is thetetrapeptide with about 0.8 equivalent NiCl₂ at pH 7.42. FIG. 17D is thetetrapeptide with about 1 equivalent NiCl₂ at pH 7.80.

FIG. 18A is the tetrapeptide at pH 7.44 with the Ala2 peak at 1.3 ppm.FIG. 18B is the tetrapeptide with about 0.3 equivalent CoCl₂ at pH 7.23.FIG. 18C is the tetrapeptide with about 0.8 equivalent CoCl₂ at pH 7.33.

FIG. 19A is the tetrapeptide at pH 7.31 with the Ala2 signal at 1.3 ppm.FIG. 19B is the tetrapeptide with about 0.5 equivalent CuSO₄ at pH 7.26.FIG. 19C is the tetrapeptide with about 1.0 equivalent CuSO₄ at pH 7.32.

FIGS. 17-19 scans were conducted at 800 MHz, 10% H₂O/90% D₂O (Ala-Meregion).

*****

The above description of the invention is intended to be illustrativeand not limiting. Various changes or modification in the embodimentsdescribed may occur to those skilled in the art. These can be madewithout departing from the spirit or scope of the invention.

1. A method of detecting or measuring an ischemic event in a patient comprising: (a) contacting a patient sample comprising full-length albumin and albumin N-terminal derivatives with an excess quantity of metal ion that binds to the N-terminus of full-length albumin, whereby albumin-metal complexes are formed, (b) partitioning the complexes from said derivatives, (c) measuring at least one of said derivatives, and (d) comparing said measured derivative to a known value, whereby the ischemic event may be detected or measured.
 2. The method of claim 1 wherein said metal is selected from the group consisting of V, As, Co, Sb, Cr. Mo, Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and Ag.
 3. The method of claim 2 wherein the metal is Ni or Co.
 4. The method of claim 1 wherein said metal of step (a) is bound to a solid support and said partitioning step (b) comprises separating said derivatives from the solid support to which the metal is bound.
 5. The method of claim 1 wherein said metal of step (a) is in solution and said partitioning step (b) comprises contacting said complexes with an antibody to the albumin-metal complex, said antibody being bound to a solid support.
 6. The method of claim 1 wherein said measuring step (c) comprises contacting said derivative with antibody to the derivative. 